DNA encoding human apoB48R: a monocyte-macrophage apolipoprotein B48 receptor gene and protein

ABSTRACT

The present invention provides an isolated DNA molecule that codes for a cell-surface binding protein in human monocytes and macrophages. In addition, an amino acid sequence derived from the nucleotide sequence is provided. The newly-identified cell-surface binding protein described herein is instrumental in the apoB-mediated cellular uptake of plasma chylomicrons and remnants and hypertriglyceridemic triglyceride-rich lipoproteins in an ApoE- and lipoprotein lipase- and heparin sulfate proteoglycan-independent pathway. The new human macrophage receptor has been cloned and uniquely, binds TGRLP via apolipoprotein B48, the marker of dietary TGRLP (apoB48R). This process rapidly converts macrophages and apoB48R-transfected Chinese hamster ovary cells in vitro into lipid-filled “foam cells,” hallmarks of atherosclerotic lesions. The apoB48R cDNA (3744 bp) encodes a novel protein with no known homologs. Its ˜3.8 kb mRNA is expressed primarily by reticuloendothelial cells. Immunohistochemical studies indicate that foam cells of human atherosclerotic lesions express the apoB48R.

CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application is a continuation-in-part and claims the benefitof priority under 35 USC §120 of U.S. patent application Ser. No.09/130,242, filed Sep. 8, 1998.

FEDERAL FUNDING LEGEND

[0002] This invention was produced in part using funds from the Federalgovernment under National Institutes of Health grant HL44480.Accordingly, the Federal government has certain rights in thisinvention.

BACKGROUND OF THE INVENTION

[0003] 1. Field of the Invention

[0004] The present invention relates generally to the fields ofmolecular biology, cardiovascular medicine and cellular nutrition. Morespecifically, the present invention relates to DNA encoding the humanmonocyte-macrophage and placental triglyceride-richlipoprotein/apolipoprotein B (apoB) receptor gene(s) and protein(s).

[0005] 2. Description of the Related Art

[0006] Hypertriglyceridemia is a common, heterogeneous disorder. Whenchylomicrons persist in the fasting state, lipid-filledmonocyte-macrophage-derived foam cells can accumulate in the spleen,liver, bone marrow, atherosclerotic lesions, and skin (Fredrickson,1978). Many, but not all, early studies (Carlson, 1972; Brunzell, 1976;Grundy, 1988; Schaefer, 1988; Austin, 1991) indicate elevated plasmatriglycerides are a risk factor for coronary heart disease andmyocardial infarction, sequelae of atherosclerosis. The possibility thattriglyceride-rich lipoproteins (hepatic as well a s dietary) areinvolved in atherosclerosis has been strengthened recently. Both theProcam study and a follow-up of the Helsinki Heart Study implicateelevated triglycerides (and therefore triglyceride-rich lipoproteins) asan important risk factor in atherosclerosis (Assmann, 1992). Havel etal. demonstrated that plasma very low density lipoprotein andintermediate density lipoprotein cholesterol levels correlated withprogression of coronary atherosclerosis disease, whereas low densitylipoprotein cholesterol level did not (Phillips, 1993). Moreover, verylow density lipoprotein-intermediate density lipoprotein particles enterthe artery wall and are found in human atherosclerotic plaques (Rapp,1994). Elevated postprandial chylomicron remnants of Sf<400 aresignificantly higher in subjects with coronary heart disease but withnormal fasting lipid levels than in matched control subjects withoutthis disease (Patsch, 1992; Weintraub, 1996). Thus, there is increasingbiochemical as well as epidemiologic evidence that the major carriers ofplasma triglycerides, very low density lipoproteins and plasmachylomicrons and their remnants, are atherogenic.

[0007] Monocytes and macrophages play a key role in atherogenesis,accounting for many lipid-filled “foam cells” in atherosclerotic lesions(Gerrity, 1981; Faggiotto, 1984). Many studies on foam cell formationhave focused on uptake of modified and oxidized low density lipoproteinby the macrophage scavenger receptor and putative oxidized low densitylipoprotein receptors (van Berkel, 1994). However, monocytes andmacrophages also take up intestinally-derived plasma chylomicrons, whichcontain apoB-48, and hepatically-derived very low density lipoprotein(apoB-100). Zilversmit and colleagues demonstrated extrahepatic uptakeof ˜40% of chylomicrons in rabbits (Ross, 1977) that was decreased byinhibition of the reticuloendothelial system (Nagata, 1987).Furthermore, studies in marmosets (a primate) and rabbits demonstratedsubstantial uptake (20-40% of total) of chylomicrons in vivo byaccessible, peripheral macrophages, particularly in bone marrow (bothanimals) and spleen (marmosets) (Hussain, 1989a, 1989b). This wouldsuggest that triglyceride-rich lipoproteins serve as a non-modified,native source of lipid for monocytes' and macrophages' nutrition in thenormal state.

[0008] Triglyceride-rich lipoproteins are involved in the pathologicalconversion of monocytes and macrophages into foam cells in humans, aprocess seen in bone marrow, spleen, etc. in types 1, 3 and 5hypertriglyceridemia (Fredrickson, 1978). Triglyceride-rich lipoproteinsare also involved in formation of monocyte-macrophage-derived foam cellsin eruptive xanthomas in untreated hypertriglyceridemic diabeticsubjects. These foam cells contained triglyceride-rich lipoprotein corelipids, triglycerides and cholesteryl esters, following chylomicronuptake (Parker, 1970).

[0009] Chylomicrons and hypertriglyceridemic-very low densitylipoproteins (including β-very low density lipoproteins) are the onlyknown native human lipoproteins, without modification, which directlycause rapid, receptor-mediated macrophage lipid accumulation in vitro,causing macrophages to resemble foam cells histologically (Gianturco,1982b, 1986a, 1986b, 1988; Brown et al., 1983; Ostlund-Lindqvist, 1983;Bersot, 1986). The lipid that accumulates in macrophages afterreceptor-mediated uptake of a lipoprotein in vitro reflects the lipidcomposition of the lipoprotein (Gianturco, 1982b; Brown et al., 1983).Therefore, as seen in vivo, triglyceride is the predominant lipid whichaccumulates initially in macrophages exposed tohypertriglyceridemic-very low density lipoproteins or chylomicrons, butcholesterol and cholesteryl esters also accumulate even in short termincubations (Gianturco, 1986a). Triglyceride-rich lipoproteins enter thearterial wall in animals (Nordesgaard, 1994) and in man (Rapp, 1994).Since one triglyceride-rich lipoprotein S_(f)>100 contains 5 times ormore cholesterol and cholesteryl esters than one low density lipoprotein(Shen, 1978), each triglyceride-rich lipoprotein that enters a monocyte,macrophage or the arterial wall is equivalent to 5 or more low densitylipoprotein particles in terms of cholesterol delivery.

[0010] A number of plausible mechanisms for the above-describedobservations exist, many involving apoE. Very low density lipoproteinsfrom hypertriglyceridemic subjects were first shown to be abnormal andpotentially atherogenic in studies which showed that very low densitylipoproteins from hypertriglyceridemic, but not from normal subjects,deliver cholesterol to cultured fibroblasts via the low densitylipoprotein receptor (Gianturco, 1978). The abnormality inhypertriglyceridemic-very low density lipoproteins is primarily in theS_(f)>60 subfraction which, in contrast to normal very low densitylipoproteins fraction S_(f)>60, contains extra apoE of an accessibleconformation that specifically binds to the low density lipoproteinreceptor; apoB of S_(f)>60 particles does not bind to the LDL receptor(Gianturco, 1982a, 1983; Bradley, 1984; Hui, 1984; Krul, 1985; Eisenberg1988). ApoE also mediates triglyceride-rich lipoprotein binding to otherwidely-distributed receptors in the low density lipoprotein receptorgene family, such as the low density lipoprotein receptor-relatedprotein/α₂-macroglobulin receptor (Beisiegel, 1989; Kowal, 1989) and avery low density lipoprotein receptor expressed primarily in heart,muscle, and adipose (Takahashi, 1992). One of these could account forapoE-mediated very low density lipoprotein uptake observed in monocytesand macrophages (Wang-Iverson, 1985).

[0011] In contrast, apoB mediates the binding of low density lipoprotein(Goldstein, 1977), intermediate density lipoproteins (S_(f)12-20), andthe predominant very low density lipoprotein in normal subjects, verylow density lipoprotein₃ (S_(f)20-60) (Bradley, 1984; Krul, 1985), theonly very low density lipoprotein subclass from normal subjects thatbinds to the low density lipoprotein receptor of fibroblasts (Gianturco,1980a, 1982a, Eisenberg, 1988) or of U937 monocytes (Sacks and Breslow,1988). The domain of apoB that binds to the low density lipoproteinreceptor is in the C-terminal portion not present in apoB-48 (Yang,1986; Milne, 1989).

[0012] Lipolysis of normal very low density lipoprotein S_(f)>60 permitsbinding of the lipolytic remnant to the low density lipoprotein receptor(Catapano, 1979; Schonfeld, 1979). Lipoprotein lipase secreted bymacrophages (Khoo, 1981) hydrolyzes very low density lipoproteins andenhances its cellular uptake (Lindquist, 1983). This facilitation mayoccur through localization of triglyceride-rich lipoproteins to membraneheparin sulfate proteoglycan (Eisenberg, 1992) and/or through binding tolow density lipoprotein receptor-related protein (Beisiegel, 1991).

[0013] The substantial and rapid uptake of triglyceride-richchylomicrons in vivo by bone marrow and spleen macrophages in marmosetsand rabbits was not accelerated by infusion of apoE (Hussain, 1989a).This is surprising, since apoE is a necessary ligand for the uptake oflarge triglyceride-rich lipoproteins by members of the low densitylipoprotein receptor gene family. Indeed, infused apoE diverted much ofthe uptake from the peripheral macrophages to the liver, suggesting thatthe observed peripheral macrophage chylomicron uptake was not mediatedby apoE and that these macrophages have an apoE-independent uptakemechanism. The rate and magnitude of triglyceride-rich chylomicronuptake by bone marrow monocytes and macrophages (20-40% of chylomicronscleared from the plasma at 20 minutes (Hussain, 1989a)) suggests thisuptake is receptor mediated. Rapid, receptor-mediated delivery ofintestinally-derived, triglyceride-enriched chylomicrons may b enecessary to assure delivery of sufficient energy and fat-solublevitamins and other essential compounds to sustain hematopoiesis. Inaddition, and in contrast to inactivation of the apoE gene, loss of apoBby homologous recombination caused embryonic lethality in the homozygousstate. ApoB is normally expressed early in yolk sak visceral endodermalcells for the synthesis of apoB-containing lipoprotein which areapparently necessary for the transport of lipids and lipid-solublevitamins to embryonic tissues.

[0014] Moreover, homologous recombinant (“knockout”) mice thatcompletely lack apoE accumulate very low density lipoprotein andchylomicron remnants in their plasma (Plump, 1992; Zhang, 1992). Thesemice develop atherosclerosis that is accelerated by high fat diets. Thelesions are characterized by monocyte-macrophage-derived foam cells, asin human lesions, demonstrating unequivocally that apoE is not necessaryfor the conversion of monocytes and macrophages into foam cells in vivo(Nakashima, 1994; Reddick, 1994). Taken together, these in vivo studiessuggest strongly the existence of an apoE-independent pathway for theuptake of triglyceride-rich lipoproteins by monocytes and macrophages,which would result in foam cell formation in hypertriglyceridemia.

[0015] In vitro evidence for an apoE- and lipoproteinlipase-independent, apoB-mediated triglyceride-rich lipoprotein receptorpathway in murine macrophages has been reported (Gianturco, 1988).Because of the potential importance of an apoE-independent,receptor-mediated pathway for triglyceride-rich lipoproteins in theformation of foam cells in human pathology, particularly inhypertriglyceridemic subjects, the human monocyte-macrophage receptorfrom the monocytic cell line THP-1 was characterized and purified andreceptor-specific antibodies were produced. Briefly, this uniqueapoE-and lipoprotein lipase-independent pathway and binding site is inmurine macrophages, human monocytes and macrophages, and in the humanmonocytic cell lines THP-1 and U937, but not in human fibroblasts orhepatoma cell lines or in Chinese hamster ovary (CHO) cells (Gianturco,1988, 1994a). Further, ligand blotting studies in bovine and porcineaortic endothelial cells also were positive. Thus, endothelial cellsspecifically bound chylomicrons followed b y hydrolysis and uptake oftheir cholesteryl esters (Fielding, 1978) and very low densitylipoproteins from hypertriglyceridemic subjects, but not from normalsubjects, delivered cholesterol to cultured endothelial cells(Gianturco, 1980).

[0016] Since the apoE-independent and lipoprotein lipase-independentreceptor also binds 13-very low density lipoproteins, but with loweraffinity, it was once referred to as a β-very low density lipoproteinreceptor (Goldstein, 1980; Gianturco, 1986a). Subsequent studies,however, demonstrated that uptake of triglyceride-rich lipoproteinsindependent of apoE was not inhibited by anti-low density lipoproteinreceptor antibodies that inhibited the low density lipoproteinreceptor-mediated uptake of rabbit β-very low density lipoproteins inthe same cells, nor did anti-low density lipoprotein receptor antibodiesbind to the candidate receptor (Gianturco, 1988). The apoE-independentreceptor differs from the low density lipoprotein receptor family or thescavenger receptor family in many properties including (1) unchangedexpression during differentiation, (2) slower intracellular liganddegradation, (3) ligand specificity, (4) apparent molecular weight ofthe candidate receptors, and (5) cellular distribution.

[0017] The prior art is deficient in the lack of the sequence of the DNAencoding for the monocyte-macrophage apoB receptor gene and protein, inthe genomic structure and chromosomal localization and in theunderstanding of its expression in the placenta, human coronary,carotid, and aortic macrophage-derived foam cells in atheroscleroticlesions and in other immune tissues including peripheral bloodleukocytes, bone marrow, spleen, tonsils and appendix. The presentinvention fulfills this longstanding need and desire in the art.

SUMMARY OF THE INVENTION

[0018] Monocyte-macrophage-derived foam cells accumulate inatherosclerotic lesions and throughout the body in some types ofhypertriglyceridemia. Uptake of plasma chylomicrons andhypertriglyceridemic triglyceride-rich lipoproteins by anapoE-independent human monocyte and macrophage receptor, distinct frompreviously-described lipoprotein receptors, may b e involved in foamcell formation in vivo. Two cell-surface membrane binding proteins(MBPs) of ˜200 and ˜235 kDa, in human monocytes and macrophages andTHP-1 monocytes and macrophages, were characterized as the likelyreceptors. It was determined that both MBPs share a common 200 kDaligand binding subunit. This ligand-binding subunit was purified andinternal tryptic peptide sequences were obtained. Receptor-specificantipeptide antibodies were generated against a 10-residue unique andunambiguous internal sequence (to which no matches were found inGenBank, Swiss Pro, etc) that binds the active receptor forms MBP200,MBP200R and MBP235. Antibodies against the C-terminal ˜47 kDa receptordomain and other domains were produced and shown to bind to all activeforms of the receptor. Overlapping partial cDNAs from a λgt10 THP-1library and from a λgt10 human placental library corresponding to thereceptor were obtained and sequenced.

[0019] The present invention shows that cell-surface MBP200 and MBP235are unique monocyte, macrophage, placental and endothelial cellreceptors for apoB in plasma chylomicrons and somehypertriglyceridemic-triglyceride-rich lipoproteins and their remnants;other apoB-containing lipoproteins also bind to the receptor withvarying, generally much lower, affinities. The present invention alsoshows that said receptors bind to apoB-48 and to the N-terminal portionof apoB-100 at or near the lipoprotein lipase binding site and not in aheparin-binding domain. Normally, the MBPs may be involved in nutritionof circulating monocytes and accessible, peripheral macrophages, e.g.bone marrow; in lipemic states, the pathway can be overwhelmed andcontribute to foam cell formation and endothelial cell dysfunction.Therefore, diminished triglyceride-rich lipoprotein uptake by thisreceptor, due either to receptor defects or to triglyceride-richlipoprotein defects leading to altered receptor affinity, may beinvolved with metabolic abnormalities associated with increased risk forcardiovascular disease, such as modest hypertriglyceridemia and small,dense low density lipoproteins (pattern B) and/or persistence ofchylomicron-derived, (i.e., apoB-48-containing) lipoproteins in thefasting state. Diminished activity of the receptor in the placenta couldresult in fetal abnormalities due to reduced delivery of dietaryfat-soluble vitamins (A, E, D) and essential fatty acids and otheressential nutrients that are carried in chylomicrons.

[0020] To clone the cDNA for MBP200R, PCR with degenerate primers wereused and a THP-1 λgt10 cDNA library to produce a 631 bp product (pcr631)(SEQ ID No. 3) which contains three peptide sequences found in aminoacid sequence from MBP200R. pcr631 was used to identify several distinctcDNA clones. One clone, THP-1 λ73-3 (SEQ ID No. 9), contains an 1851 bpinsert with a 1381 bp open reading frame (ORF) and 470 bp untranslatedregion including the stop codon, the polyadenylation signal and thepoly-A tail. PCR on a 5′ stretch human placenta λgt10 cDNA library usingantisense primers derived from the 5′ end of THP-1 λ73-3 resulted in a1466 bp clone with an open reading frame that overlapped the openreading frame of THP-1 λ73-3 (pcr1466) (SEQ ID No. 8) resulting in a3071 bp sequence with a 2601 bp open reading frame.Glutathione-S-transferase fusion proteins were expressed using thepcr631, THP-1 λ73-3, and pcr1466 pGEX constructs. Polyclonal antibodieswere produced to each protein domain. Immunoblots demonstrate that theantibodies specifically recognize the GST-fusion products and allreceptor activities (MBP200, MBP235 and MBP200R). Additional 5′ sequenceobtained by PCR of the human placenta λgt10 cDNA library with antisenseprimers from the 5′ end of pcr1466 resulted in a 75 bp clone (pcr751)(SEQ ID No. 7) that contained a 10 bp untranslated 5′ end, a Kozakconsensus start sequence and the initial ATG start codon. The sequencesobtained from multiple clones from THP-1 monocyte genomic DNA and thecDNA library result in 3744 bases of cDNA sequence (SEQ ID No. 1) withan open reading frame of 3264 b p encoding a 1088 residue protein forthe human monocyte apoB48 receptor. Northern analysis of THP-1s, humanplacenta, bone marrow, peripheral blood leukocytes, spleen, tonsils,appendix, and lymph node reveal a messenger RNA of approximately 3.8 kb,indicating the complete cDNA sequence has been determined. A full-lengthcDNA was constructed in a pCDNA vector. Chinese hamster ovary (CHO)cells transfected with the vector containing the receptor cDNA, incontrast to the pCDNA vector alone, expressed full receptor activity asdetermined by rapid, high affinity binding and uptake of fluorescentDiI-labeled trypsinized VLDL and, in stably transfected CHOs, by rapidcellular triglyceride mass accumulation and by the rapid (˜1.5 hour)accumulation of cytoplasmic lipid droplets visualized by lightmicroscopy after staining with the neutral lipid Oil Red 0, afterincubation with chylomicrons containing apoB48 as the B species,HTG-VLDL and tryp VLDL but not with normal VLDL or LDL.

[0021] One object of the present invention is to provide a n isolatedDNA molecule encoding a monocyte-macrophage cell surface apoB48R bindingprotein selected from the group consisting of: (a) a DNA moleculecomprising a sequence SEQ ID No. 1 and which encodes themonocyte-macrophage cell surface apoB48R binding protein (the apoBreceptor) (SEQ ID No. 2) or a portion of the monocyte-macrophage cellsurface apoB48R binding protein; and (b) a DNA molecule differing fromthe DNA molecule of (a) in codon sequence due to the degeneracy of thegenetic code, and which encodes the monocyte-macrophage cell surfaceapoB48R binding protein (SEQ ID No. 2) or a portion of themonocyte-macrophage cell surface apoB48R binding protein. Embodiments ofthis object of the invention include provisions for a vector containingthe isolated DNA molecule encoding a monocyte-macrophage cell surfaceapoB48R binding protein and regulatory elements necessary for expressionof said isolated DNA molecule in a cell, the vector adapted forexpression in a recombinant cell, as well as a host cell containing thevector.

[0022] An additional object of the present invention is to provide avector comprising an isolated DNA for a monocyte-macrophage cell surfaceapoB48R GST fusion binding protein having the sequence SEQ ID No. 2 orportions thereof.

[0023] A further object of the present invention is to provide a methodof cell-specific delivery of therapeutic compounds to human monocytes,macrophages, other reticuloendothelial cells that express the receptoror embryos comprising the steps of: providing a peptide or antibody(s)having the ability to bind to an isolated monocyte-macrophage cellsurface apoB48R binding protein having the sequence SEQ ID No.2, or aportion of said sequence or comprising a related protein of the samegene family; and incorporating the peptide into liposomes containingsaid therapeutic compound. Yet another method of cell-specific deliverymay utilize a receptor-specific antibody or an antibody fragment (Fab)that binds to a n isolated monocyte-macrophage cell surface apoB48Rbinding protein having the sequence SEQ ID No. 2, or a portion of thesequence, or comprising a related protein of the same gene family; andincorporating the antibody into liposomes.

[0024] Yet another object of the present invention is to provide amethod of inhibiting foam cell formation and increased monocyte adhesionto endothelial cells, comprising the step of treating amonocyte-macrophage with an agent which binds an isolatedmonocyte-macrophage cell surface apoB48 receptor protein having thesequence SEQ ID No.2, thereby blocking or inhibiting binding ofapoB-containing lipoproteins to the receptor. Gene therapy such asadenoviral delivery of the receptor proteins of the present invention toLDL-receptor deficient subjects is also contemplated.

[0025] Another object of the present invention is to provide delivery ofthe novel sequences disclosed herein, e.g., in an adenoviral vector, tothe liver or elsewhere, for the purpose of correcting metabolic defectsthat cause abnormal accumulation of apoB-containing lipoproteins in theplasma.

[0026] Yet another object of the present invention is to provide amethod of evaluating an individual at risk for cardiovascular disease,comprising the steps of: (a) extracting a sample ofmonocytes-macrophages and triglyceride-rich lipoproteins from the plasmaof the individual and from a control individual not considered at riskfor cardiovascular disease; and (b) comparing the binding affinity(K_(d)) of the apoB receptor of the monocytes-macrophages fortriglyceride-rich lipoproteins between the individual at risk and thecontrol individual, whereby a difference in the binding affinity betweenthe individual at risk and the control individual is indicative of analteration in either or both the apoB cell-surface receptor protein andtriglyceride-rich lipoproteins, and the alteration in the apoBcell-surface receptor protein or triglyceride-rich lipoproteins isindicative of dyslipidemias, abnormal postprandial triglyceridemetabolism or Pattern B phenotype in the individual at risk.Alternatively, one may compare the rapid, receptor mediatedmonocyte-macrophage lipid accumulation induced by standardized tryp VLDLvs. normal TGRLP vs the patients TGRLP by quantifying lipid mass changesand by Oil Red 0 staining.

[0027] This objective may also be accomplished by performing a Westernblot analysis on proteins of the monocytes-macrophages using an antibodydirected towards the protein of SEQ ID No. 2, or fragments thereof, oralternatively performing a Northern blot analysis on RNAs of themonocytes-macrophages using a DNA probe selected from the groupconsisting of SEQ ID No. 1, SEQ ID No. 3, SEQ ID No. 7, SEQ ID No. 8 andSEQ ID No. 9, or fragments thereof, whereby a difference in themigration or mobility of the proteins and/or RNAs between the individualat risk and the control individual is indicative of an alteration in theapoB cell-surface receptor protein, and the alteration in the apoBcell-surface receptor protein is indicative of dyslipidemias, abnormalpostprandial triglyceride metabolism or Pattern B phenotype in theindividual at risk.

[0028] As atherosclerotic plaques are enriched with ApoB48-receptorexpressing cells, the instant invention may also be directed towarddelivery of therapeutic agents to atherosclerotic plaques. These agentsmay include label to localize said plaques, inhibitors of apoB48receptor to inhibit further development of the plaques, or agentsdesigned to eliminate the plaques.

[0029] Other and further aspects, features, and advantages of thepresent invention will be apparent from the following description of thepresently preferred embodiments of the invention given for the purposeof disclosure.

BRIEF DESCRIPTION OF THE DRAWINGS

[0030] So that the matter in which the above-recited features,advantages and objects of the invention are attained and can beunderstood in detail, more particular descriptions of the invention maybe had by reference to certain embodiments which are illustrated in theappended drawings. These drawings form a part of the specification. Itis to be noted, however, that the appended drawings illustrate preferredembodiments of the invention and therefore are not to be consideredlimiting in their scope.

[0031]FIG. 1 shows a 2-dimensional SDS-PAGE of MBP200 and MBP235 beforeand after reduction. Detergent extracts of THP-1 monocytes wereelectrophoresed in the first dimension in a minigel without reduction.One cm strips containing the MBPs were removed and treated with buffer(panel A) or reductant, 2% 2-mercaptoethanol, (panel B), placedlengthwise on a second 5% slab minigel, electrophoresed and transferredto nitrocellulose for ligand binding analysis. Panel A illustrates thatwithout reduction MBP200 and MBP235 maintain their distinct mobilities(seen on the diagonal), whereas after reduction (R) in the seconddimension, both activities now have identical Rfs that are differentfrom their original Rfs. The reduced form of both receptor proteins thatretain ligand binding activity was called MBP200R. The single lane onthe far left of each 2-D gel contains internal prestained markers, atapproximately 200 kDa and 97 kDa.

[0032]FIG. 2 shows (2A) ligand binding of MBPs by ligand blot analysis.Concentrations of the hypertriglyceridemic-very low density lipoproteinsare given below each lane before (non-reduced) and after (reduced)treatment with 2-mercaptoethanol. Very low density lipoprotein bindingwas detected by apoB antibodies followed by IgG-specific enzyme-linkedantibody and calorimetric substrate. Densitometry and quantification ofthe images utilized ImageQuant software. FIG. 2B shows the saturationbinding of hypertriglyceridemic-very low density lipoprotein to MBP235,MBP200 and MBP200R. The amount of very low density lipoprotein bound toMBP200, MBP235 and MBP200R activities in FIG. 2A was determined bydensitometry of each MBP region in the ligand blots using a calibrationcurve generated from known amounts of the same very low densitylipoprotein applied to nitrocellulose and quantified with a purifiedanti-apoB antibody. The amount of ligand bound to each MBP region,expressed as ng very low density lipoprotein bound, was plotted as afunction of the amount of very low density lipoprotein to which thenitrocellulose was exposed. The broken line represents the calculatedsum of binding of the very low density lipoprotein to MBP200 and MBP235at each level of very low density lipoprotein. FIG. 2C shows the datafrom FIG. 2B after transformation plotted by the method of Scatchard todetermine the K_(d) for each of the MBPs, expressed as μg/ml.

[0033]FIG. 3 shows a model of the relationship between MBP235 and MBP200and how MBP200R is generated from MBP200 and MBP235 by treatment withreducing agents such as 2-mercaptoethanol.

[0034]FIG. 4 shows that the ligand binding activity and antipeptideimmunoreactivity are coincident before and after reduction of the MBPs,demonstrating that each MBP contains the 10-residue unique sequenceobtained by microsequence data from peptides from purified MBP200R.C=preimmune sera at the appropriate concentration as control. Lanes 1-6contain two levels of the THP-1 detergent extracts: lanes 1, 3 and 5 area 1:3 and lanes 2, 4 and 6 are 1:5 dilution. Lanes 3, 4 and 8 are at1:50 and lane 9 is at 1:100 dilution of the antipeptide antibody. Ligandbinding activity was visualized by incubation withhypertriglyceridemic-very low density lipoproteins for 2 hours at roomtemperature and detected with anti-apoB antibodies. Primary antibodybinding was then detected with Anti-IgG alkaline phosphatase conjugatedantibodies.

[0035]FIG. 5 shows that anti-apoB antibodies block binding oftriglyceride-rich lipoproteins to MBP200 and MBP235. Biotinylated verylow density lipoproteins were incubated for 0.5 hour with anti-apoBantibodies, lane 2, or with preimmune serum, lane 1, prior to ligandblotting for 1.5 hours. Biotinylated very low density lipoproteinbinding was detected with streptavidin-alkaline phosphatase. The blotswere imaged, then quantitated with ImageQuant. The relative totalbinding to the MBPs with the specific apoB antisera and the preimmunesera are as indicated. The insert contains the imaged lanes from theligand blot.

[0036]FIG. 6 shows the analysis of THP-1 monocytes by FACS. Antipeptideantibodies are shown to bind to the surface of THP-1 monocytes using twoconcentrations of the primary antibody. Goat(Fab′)₂ rabbit anti-IgG(H+L)-FITC only (used as the negative control) and the alpha chain ofthe monocyte integrin VLA-4 was used as a known positive control.

[0037]FIG. 7 shows the amino acid sequence derived from the 631 basepair PCR product. The carboxyterminal sequence (shaded) represents thepeptide sequence used to produce the initial degenerate oligonucleotideprimer and is from the same peptide used to develop the anti-peptideantibodies and used for surface labeling of the THP-1 monocytes. Theunderlined peptides represent sequences found by tryptic peptide mappingand microsequence analysis of MBP200R.

[0038]FIG. 8 shows that THP-1 monocytes contain mRNA coding for MBP200R.RT-PCR using primer pairs from t h e PCR631/MBP200R sequence and mRNAfrom THP-1 monocytes produced a RT-PCR product of the correct size. Theethidium bromide stained gel (inverse image) of the RT-PCR products isshown. Lane 1: 572 bp product using primers 53-72, sense and 624-604,antisense; Lane 2: Glucose-6-phosphate dehydrogenase (GPDH) controlproduct; Lane 3: MW ladder; Lane 4: second round product generated withinternal second antisense primer, 490 bp.

[0039]FIG. 9 shows the restriction mapping of the 572 bp RT-PCR productdescribed in FIG. 8 (inverse image). Lane 1: 572 bp product; Lane 2:EcoRI control, no digestion; Lane 3: BamH1; Lane 4: MW ladder 50 bp,100, 200, 300, 400, 500, 700, 1000, 1500 and 2000 bp; Lane 5: PstI; Lane6: HindIII; Lane 7: XbaI.

[0040]FIG. 10 shows that monospecific rabbit anti-human apoB IgGsspecifically inhibit the binding of HTG-VLDL to the monocyte TGRLPreceptor (MBP200 or MBP235). THP-1 monocyte aqueous phase extracts wereelectrophoresed and transferred to nitrocellulose (˜100 μg/lane).Biotinylated HTG-VLDL S_(f)100-400 (0.5 μg/ml) was preincubated withbuffer (lane 1) or with 2 levels of anti-apoB (rabbit 1325) 40 μg/mLlane 2; 400 μg/ml lane 3; or two levels of preimmune (rabbit 1325) IgG90 μg/mL, lane 4 and 400 μg/ml, lane 5. Lipoproteins and IgGs werepreincubated for 30 minutes and then incubated with the nitrocellulosestrips for 3 hours at 4° C. After extensive washing, bound lipoproteinwas detected with streptavidin linked to alkaline phosphatase followedby colorimetric substrates (the digitized image is shown in (FIG. 10A)and quantified by scanning densitometry (FIG. 10B) using two-dimensionarea integration and illustrated as VLDL binding in densitometric units(pixels).

[0041]FIG. 11 shows that anti-apoB, but not anti-apoE, anti-apoCIII, ornonimmune IgG, inhibits the binding of HTG-VLDL S_(f)100-400 to MBP200or MBP235. THP-1 monocyte aqueous phase extracts were electrophoresedand transferred to nitrocellulose, blocked, and incubated withbiotinylated HTG-VLDL and the indicated antibodies at 4° C. Lane 1,buffer; lane 2, 3 mg/ml anti-apoB (1325); lane 3, 2.4 mg/ml nonimmuneIgG; lane 4, 2.3 mg/ml anti-apoE; lane 5, 2 mg/ml anti-apoCIII. The IgGswere preincubated with biotinylated HTG-VLDL (2.5 μg/ml) for 30 minutesat 4° C. and then were incubated with the nitrocellulose strips for 3hours at 4° C. Lipoprotein binding was visualized (FIG. 11A) andquantified (FIG. 11B) by densitometry as described in FIG. 10.

[0042]FIG. 12 shows that anti-apoB IgG, but not nonimmune IgGs, inhibitthe binding of ¹²⁵I-HTG-VLDL to THP-1 macrophages (top panel) but not tohuman fibroblasts with upregulated LDL receptors. (bottom panel) THP-1monocyte-macrophages one day after adherence were grown as described.Duplicate dishes of cells and no cells for controls were incubated with¹²⁵I-HTG-VLDL S_(f)100-400, 5 μg/ml, alone (none) or in the presence ofa 30-fold excess of unlabeled HTG-VLDL (self), in the presence ofaffinity-purified sheep anti-apoB IgG (anti-apoB), or the equivalentlevel of sheep nonimmune IgG (Non-1 mm) at 4° C. for 16 hours and thenincubated with precooled, washed cells for 1.5 hours at 4° C. Afterextensive washing, the cells were dissolved in 0.1 NaOH fordetermination of bound ¹²⁵I-HTG-VLDL as described. Values represent theaverage of duplicate dishes corrected for background by subtracting theaverages of the no-cell controls and are expressed in terms of percentof control, that is percent of the uninhibited activity (100%). Specificbinding activity for ¹²⁵1-HTG-VLDL, 5 μg/ml was 24 ng/mg THP-1 cellprotein which represented 100% uninhibited activity. (top panel) Humanskin fibroblasts were grown to 75% confluency and preincubated with DMEcontaining 5% lipoprotein-deficient serum for 36 h to induce the LDLreceptor. The binding and competition protocols were identical to thosefor the THP-1 cells as described above. Specific binding activity for¹²⁵I-HTG-VLDL, 5 μg/ml was 237 ng/mg fibroblast cell protein whichrepresented 100% uninhibited activity.

[0043]FIG. 13 shows the effects of lactoferrin and heparin on binding ofHTG-VLDL to MBP200 and MBP235 (these agents fail to inhibit or enhancebinding). THP-1 monocyte aqueous phase extracts were electrophoresed andtransferred to nitrocellulose. The nitrocellulose strips were incubatedfor 4 hours at 4° C. with 0.5 μg biotinylated HTG-VLDL/ml in the absence(lane 1) or in the presence of lactoferrin at 50 μg protein/ml (lane 2)or 500 μg protein/ml (lane 3); heparin at 10 U/ml (lane 4) and 100 U/ml(lane 5); or unlabeled HTG-VLDL at 25 μg/ml (lane 6) or 5 μg/ml (lane7). Biotinylated HTG-VLDL binding was detected with streptavidin linkedalkaline phosphatase (digitized image, (FIG. 13A) and quantified bydensitometry (FIG. 13B).

[0044]FIG. 14 shows that lipoprotein lipase inhibits the binding ofHTG-VLDL to MBP200 and MBP235. THP-1 monocyte aqueous phase extractswere electrophoresed and transferred to nitrocellulose, blocked, andincubated at 4° C. with biotinylated HTG-VLDL S_(f)100-400 (3 μg ofprotein/ml) in the absence (lane 1) or in the presence of lipoproteinlipase (0.2 μg/ml, lane 2; 2.0 μg/ml, lane 3; 20 μg/ml, lane 4) or inthe presence of bovine serum albumin (0.2 μg/ml, lane 5; 2.0 μg/ml, lane6; 20 μg/ml, lane 7). Binding was detected by incubation withstreptavidin-alkaline phosphatase and the image digitized (FIG. 14A) andquantified (FIG. 14B) by densitometry.

[0045]FIG. 15 shows immunoblots that demonstrate that plasmachylomicrons of S_(f)>1100 contain apoB-48 but not apoB-100. Plasma wasisolated from a hypertriglyceridemic subject 4 hours after astandardized test fat meal. Total chylomicrons S_(f)>400 weresubfractionated through a salt gradient into 3 subclasses: S_(f)>3200(CM I), S_(f)1100-3200 (CM II), S_(f)400-1100 (CM III). These wereelectrophoresed at two levels of each (1 and 2 μg total apoprotein/lane)on a 4-20% SDS-PAGE, transferred to nitrocellulose, and probed for apoB(above the line) and apoE (below the line). CM I, lanes 1 and 2; CM II,lanes 3 and 4; and CM III, lanes 5 and 6; and controlhypertriglyceridemic VLDL S_(f)100-400, containing apoB-100 and apoB-48and apoE, in lane 7; lane M=prestained protein molecular weight markers.

[0046]FIG. 16 shows the binding of chylomicron subspecies to MBP200 andMBP235 (odd lanes) or to the partially purified LDL receptor (evenlanes). THP-1 monocyte aqueous phase extracts and the partially purifiedbovine LDL receptor were electrophoresed in alternating lanes,transferred to nitrocellulose, blocked, and incubated with chylomicronsubfractions (10 μg/ml). CM I (S_(f)>3200) were incubated with lanes 1,2; CM II (S_(f)1100-3200) with lanes 3, 4; and CM III (S_(f)400-1100)with lanes 5, 6. Binding was detected with a polyclonal anti-apoBantibody followed by a second antibody linked to alkaline phosphatase.The sharp band in lanes with THP-1 extracts that migrates between MBP200and the LDL receptor is a nonspecific lipoprotein binding proteinapparent in some but not all ligand blots.

[0047]FIG. 17 shows that anti-apoB antibodies specifically block thebinding of chylomicrons S_(f)1100-3200 (CM II) that contain apoB-48 asthe only apoB species to MBP200 and MBP235. Thus, this receptor is alsoreferred to as the apoB48 receptor. THP-1 aqueous phase extracts wereelectrophoresed and transferred to nitrocellulose. Prior to incubationwith the nitrocellulose strips for 3 hours at 4° C., biotinylated CM IIwere preincubated at 4° C. for 30 minutes with buffer (lane 1), withanti-apoB (rabbit 1325) (lane 2), or with an equivalent level ofnonimmune IgG (lane 3). Chylomicron binding was visualized afterincubation with streptavidin-linked alkaline phosphatase, digitized(FIG. 17A) and quantified (FIG. 17B) by scanning densitometry.

[0048]FIG. 18 shows the expression of the 3.8 kb TGRLP/ApoB ReceptormRNA in human placenta and THP-1 monocytes-macrophages.

[0049]FIG. 19 shows the expression of TGRLP/ApoB Receptor mRNA in humanimmune tissues, including spleen, lymph node, thymus, appendix, bloodleukocytes and bone marrow.

[0050]FIG. 20 shows the relative positions of the overlapping clonesused to characterize the full-length human monocyte TGRLP/ApoB ReceptorcDNA.

[0051]FIG. 21 shows the nucleotide sequence and derived amino acidsequence of the THP-1 monocyte apoB48 receptor cDNA. Nucleotides arenumbered at the right on top with every 10 bp indicated by a “V”. Theamino acid residues are also numbered at the right with 20 residues perline. The ATG start site codon is preceded by 10 bp; a Kozak startsequence is at bp 5-14. The entire cDNA sequence is 3744 bp. The derivedprotein length is 1088 residues, with the TGA stop codon at bp 3275. The10-residue unambiguous amino acid sequence (amino acids 910-919)determined from microsequence analysis of the tryptic hydrolysis ofpurified apoB48 receptor and used to design the degenerateoligonucleotide primers is indicated with a double underline, as are twoother tryptic peptides likewise identified. The single underlined 23amino acid segment (amino acids 751-773) is the putative transmembranedomain. Cysteine residues are bold and underlined (C); potentialcoiled-coil interactive domains are bold and italicized (amino acid155-189; amino acid 473-486; and amino acid 520-533). There are twopotential polyadenylation signals beginning at bp 3700 and bp 3708. Bothstrands of the cDNA were sequenced; additional sequencing runs werecarried out in some cases in regions of high CG content.

[0052]FIG. 22 shows the localization of the apoB48 receptor gene tochromosome 16p11 by fluorescence in situ hybridization (FISH). HumanapoB48R cDNA (1-2614) was labeled with Cy3-dUTP by nick translation andhybridized to normal human metaphase and interphase nuclei fromphytohaemagglutinin-stimulated blood lymphocytes. Chromosomes werecounterstained with DAPI and the fluorescence images captured withdigitized image microscopy. DAPI-stained chromosomes (blue) withCy3-labeled hybridization signal (red) are paired with the black andwhite images of the DAPI-stained chromosomes pair for chromosomeidentification and for hybridization location.

[0053]FIG. 23 shows PCR screening of a multiple human tissue cDNA panel.A multiple tissue cDNA panel (MTC™, Clontech) containing normalized,first strand cDNA from (in order from left to right) brain, heart,kidney, liver, lung, pancreas, placenta, skeletal muscle, positivecontrol (for combined tissues), no DNA control, and GPDH control werePCR-amplified using apoB48 receptor sequence specific primersoverlapping introns 2 and 3 (Table 2) to ensure that only mRNA speciesare reflected. The expected 455 bp product was found in all but skeletalmuscle. The level of apoB48R cDNA appeared greatest in lung and placenta(seen after 25 cycles) and lowest in brain and heart (seen only after 35cycles). Control PCR analyses of the individual tissues for the GPDHcDNA verified normalization of tissues and reflect relative mRNAabundance in that tissue.

[0054]FIG. 24 shows western and ligand blotting activity detected inapoB48R transfected CHO-K1s. A ligand blot (Gianturco, S. H., et al.,1998) is shown on the left and western blot on the right of detergentextracts of, from outer to inner lanes, THP-1 monocytes as a positivecontrol, CHO-K1's transfected with the empty pcDNA expression vector,and CHO-K1's transfected with the apoB48 receptor minigene that containsthe first intron. Tryp-VLDL S_(f) 100-400 (Gianturco, S. H., et al.,1986) served as the ligand, detected with an anti-apoB antibody andalkaline phosphatase-linked IgG second antibody. The apoB48R protein wasvisualized using a polyclonal antibody prepared against an apoB48R-GSTfusion protein containing amino acids 223 to 710. The THP-1 extractsillustrate the two reported forms of the receptor, the ˜200 kDa ligandbinding species and a ˜235 kDa form that contains both the ligandbinding ˜200 kD species and a noncovalently bound protein, possibly achaperone (Ramprasad, M. P. et al., 1995). Neither ligand blottingactivity nor receptor immunoreactivity is detected in thevector-transfected negative controls. In contrast, theapoB48R-transfected CHOs exhibit both ligand blotting activity andapoB48R immunoreactivity with identical electrophoretic mobilities. Theapparent molecular weight of the transfected apoB48R is slightly less(190 kD) than that of the 200 kDa activity in THP-1s. This is likely dueto processing differences in CHO cells versus humanmonocyte-macrophages. Of note, however, the apparent molecular weight ofthe apoB48R in the CHOs is approximately twice that predicted by thecDNA.

[0055]FIGS. 25A and 25B show expression of the apoB48R: FIG. 25A showsTG accumulation in apoB48R-transfected CHO-K1 cells, but not in controlcells, incubated with tryp-VLDL S_(f) 100-400. G418-selected, stabletransfected CHOs were grown in 6 well tissue culture dishes. BothapoB48R-transfected and vector with inverted insert-transfected cellswere incubated with tryp-VLDL S_(f) 100-400 (Gianturco, S. H., et al.1986) at the concentrations indicated for 4 hrs at 37° C. and the cellsprocessed to measure TG mass a s previously reported (Gianturco, S. H.,et al., 1988). The upper curve (closed squares; apoB48R transfected)indicates a rapid, curvilinear accumulation of TG with increasing levelsof tryp-VLDL S_(f) 100-400 reflective of the receptor-mediated uptake asseen in monocyte-macrophages (Gianturco, S.H., et al., 1994C), while thelower curve (open squares; pcDNA 3.1 vector plus inverted inserttransfected) demonstrates a low affinity, linear accumulation, as seenin cells where no apoB48R activity is present, representing lowaffinity, nonspecific uptake. Values are averages from duplicate dishesthat differ by <10%; This is one representative experiment of fourexperiments with the full length cDNA and four with the cDNA containingthe first intron. FIG. 25B shows transfection of the apoB48R and itsrole in uptake of TGRLP and cellular TG accumulation. ApoB48R wastransfected into CHO K1 cells and selected with G418 as described inFIG. 25A. The transfected cells were placed in 6-well plates and testedfor their ability to accumulate TG when exposed to different TGRLP as ameasure of lipoprotein-specific receptor function, as previouslyreported (Gianturco, S. H, et al., 1994c). After a 2 hour incubation at37° C., the transfected cells were washed in cold PBS to remove excessTGRLP, the cellular lipid extracted with hexanes:isopropanol (3:2, v/v)and the TG level measured enzymatically. The bar graph indicates theamount of TG that accumulated per mg of cell protein relative to abuffer control (the buffer level was subtracted from the level for eachindicated lipoprotein). This value was also corrected for nonspecificbinding of TGRLP to the well by subtracting a no-cell control value ateach level of TGRLP used. Like human THP-1 macrophages, theapoB48R-transfected cells accumulate TG when exposed to apoB48-onlycontaining chylomicrons S_(f) 1100-3200 (CMII) and to the model ligand,tryp-VLDL S_(f) 100-400 (t-V1) but not to VLDL S_(f) 100-400 from asubject with normal plasma TG levels (N-V1). The experiment was repeatedtwice with identical results. Concurrent experiments withreceptor-negative, vector-only transfected CHO-K1s detected nosignificant TG accumulation under the same experimental conditions (datanot shown).

[0056]FIGS. 26A and 26B show oil-red-O staining of neutral lipid, whichwas used to assess receptor activity in transfected cells. In FIG. 26A,ApoB48R-transfected CHO-KI cells show massive accumulation of lipidafter a three hour incubation at 37° C. with chylomicrons S_(f)>400 at100 μg TG/ml in RPMI medium with no other serum components, asdemonstrated by the Oil Red O-positive cytoplasmic lipid droplets in thecells. These chylomicrons contain apoB48 as their only apoB species,consistent with the ligand specificity of this receptor. FIG. 26Bdemonstrates that vector only-transfected cells showed no appreciableOil-red-O staining after incubation under identical conditions.Receptor- and vector-transfected cells treated with buffer also showedno lipid staining inclusions (not shown).

[0057]FIGS. 27A, 27B, 27C, and 27D show immunohistochemicaldemonstration of apoB48R in human carotid artery atheroscleroticlesions. Serial sections of carotid arteries obtained by endarterectomywere processed as described. Rabbit polyclonal antibodies producedagainst an apoB48R-domain specific (amino acid 223-710) GST-fusionprotein were used to determine apB48R expression and location. Allmagnifications are 100×, except for FIG. 27C, which is 80×. FIG. 27Ashows a hematoxylin-stained section of an advanced lesion with the lumenoriented toward the top and a lipid core surrounded by foam cells seento the right and center. FIG. 27B shows staining with amacrophage-specific monoclonal antibody (HAM56), which identifies themacrophage-derived foam cells on the edge of the acellular lipid core.FIG. 27C is a negative control, which used preimmune IgG from the samerabbits used to generate the anti-apoB48R-specific antibodies. In FIG.27D, anti-apoB48R antibodies indicate apoB48R-expression by lesionmacrophages and foam cells.

DETAILED DESCRIPTION OF THE INVENTION

[0058] Two major triglyceride-rich lipoprotein membrane bindingactivities with apparent molecular weights of approximately 200 andapproximately 235 kDa (MBP200 and MBP235) were identified by ligandblotting analysis in both normal human blood-borne monocyte-macrophagesand the long term human THP-1 and U937 monocytes and macrophages. MBP200and MBP235 are cell surface proteins that share a common backbone(MBP200) containing the ligand binding domain. MBP235 is comprised ofMBP200 plus one (or more) small subunit(s) of ˜35 kDa apparent totalmass (as determined by mobilities on SDS-PAGE) that associate(s)noncovalently with MBP200, does not inhibit triglyceride-richlipoprotein binding, and is immunochemically distinct from thereceptor-associated protein (RAP), a 39 kDa protein that modulatesligand binding to low density lipoprotein receptor-related protein andother low density lipoprotein receptor family members (Strickland,1990).

[0059] Amino acid sequence data obtained from tryptic peptides frompurified MBP200R, the reduced ligand-binding subunit of the receptor,had no matches in gene and protein databases, indicating that MBP200 isa unique protein. The sequence data was used to produce antipeptideantibodies that were then found to bind solely to the MBP200, MBP235,and MBP200R receptors, thereby confirming that MBP200 is a uniqueprotein. The receptor-specific antibodies also confirmed the structure,chemistry and relationships of MBPs obtained initially by biochemicaland ligand blotting analyses.

[0060] The specific, apoE-independent binding of plasma chylomicrons andother apoB-containing lipoproteins to this monocyte-macrophage receptor,coupled with in vivo studies, suggests a role of this pathway in thenutrition of circulating monocytes and accessible macrophages, such asin bone marrow in the postprandial state. Moreover, anti-apoB blockingstudies indicate this previously-undiscovered human monocyte-macrophagereceptor binds triglyceride-rich lipoproteins via apoB. In addition,apoB-48 appears to be sufficient, and neither apoB-100 nor apoE isnecessary for binding. This also suggests a new role for apoB-48; i.e.,targeting plasma chylomicrons and their remnants to accessible monocytesand macrophages for uptake while still triglyceride-rich, beforereaching the triglyceride-depleted, cholesteryl ester- and apoE-enrichedremnant that is targeted by apoE to the liver for uptake by hepaticreceptors which bind lipoproteins via apoE. A defect in thismonocyte-macrophage receptor or in its ligands could result in aberranttriglyceride-rich lipoprotein metabolism (rerouting of chylomicrons forlipolysis and eventual liver uptake) that would lead to delayedchylomicron clearance and abnormal persistence of chylomicron remnantsand/or small, dense low density lipoprotein. This condition has beentermed Pattern B phenotype.

[0061] The Pattern B phenotype is expressed primarily in adults, isassociated with increased risk of cardiovascular disease, and isinherited in an autosomal dominant or codominant manner with varyingpolygenic effects, including lipoprotein lipase deficiency, insulinresistance, apo CIII, and an as-yet, unidentified gene defect(s)(Krauss, 1994). Since candidate mechanisms for this phenotype includealtered triglyceride-rich lipoprotein metabolism and clearance, themonocyte-macrophage receptor of the present invention is a candidategene for one contributing cause of this pattern. The potential role ofapo CIII in modulating the binding of triglyceride-rich lipoproteins tothis receptor fits with the observation that CIII-enrichedtriglyceride-rich lipoproteins are found in Pattern B and thattransgenic mice that overexpress CIII or CII are hypertriglyceridemic.The apoCs (especially CIII) could interfere with apoE-mediated uptakemechanisms and also mask the apoB domain(s) that bind to MBP200.Identification of genes related to this phenotype allows identificationof subjects before the phenotype is expressed. As subjects with thisphenotype are extremely responsive to changes in dietary fat (Krauss,1994), early identification permits dietary intervention so as to delayatherogenic changes.

[0062] The experiments leading to the present invention addressedseveral interactions of triglyceride-rich lipoproteins with monocytesand macrophages and their relation to lipoprotein metabolism and foamcell formation. Such interactions include a) whether MBP200 and MBP235are chylomicron/apoB-48 receptors; b) the molecular structure andfunction of MBP200; c) the structure and function of the smallsubunit(s) in MBP235 (such as chaperone(s)); d) whether MBP200 andMBP235 are restricted to monocytes, macrophages and endothelial cells;e) the receptor binding domains in apoB; and f) whether apoCIII and apoEmodulate receptor binding.

[0063] The present invention provides a composition of matter comprisingisolated DNA molecules encoding overlapping domains of themonocyte-macrophage and placental cell-surface binding protein and thefull-length cDNA construct selected from the group consisting of: (a) aDNA molecule comprising a sequence SEQ ID No. 1 and which encodes thefull-length cDNA of said monocyte-macrophage cell surface apoB48Rbinding protein (SEQ ID No. 2) or a portion of said monocyte-macrophagecell surface apoB48R binding protein domain of the sequence of themonocyte macrophage binding protein; and (b) a DNA molecule differingfrom the DNA molecule of (a) in codon sequence due to the degeneracy ofthe genetic code, and which encodes said monocyte-macrophage cellsurface apoB48R binding protein (SEQ ID No. 2) or a portion of saidmonocyte-macrophage cell surface apoB48R binding protein; an isolatedmonocyte-macrophage cell surface apoB48R binding protein having thesequence SEQ ID No. 2; a method of cell-specific delivery of therapeuticcompounds to human monocytes or macrophages, comprising the steps of:providing a peptide or antibody having the ability to bind to anisolated monocyte-macrophage cell surface apoB48R binding protein havingthe sequence SEQ ID No.2, or a portion of said sequence; andincorporating said peptide or antibody into liposomes containing saidtherapeutic compound or directly linking said peptide or antibody totherapeutic compound; and a method of inhibiting foam cell formation andincreased monocyte adhesion to endothelial cells, comprising the step oftreating a monocyte-macrophage with an agent which binds an isolatedmonocyte-macrophage cell surface apoB48R binding protein having thesequence SEQ ID No.2.

[0064] Further provided are methods of evaluating an individual at riskfor cardiovascular disease using the compositions of matter providedherein for examining either the apoB receptor-ligand interaction, or theRNA and/or protein corresponding to apoB in an individual at risk ascompared to a control individual, to determine the presence of anyabnormalities in the apoB receptor of the individual at risk.

[0065] In accordance with the present invention, there may be employedconventional molecular biology, microbiology, and recombinant DNAtechniques within the skill of the art. Such techniques are explainedfully in the literature. See, e.g., Sambrook, Fritsch & Maniatis,“Molecular Cloning: A Laboratory Manual (1982); “DNA Cloning: APractical Approach,” Volumes I and II (D.N. Glover ed. 1985);“Oligonucleotide Synthesis” (M. J. Gait ed. 1984); “Nucleic AcidHybridization” [B. D. Hames & S. J. Higgins eds. (1985)]; “Transcriptionand Translation” [B. D. Hames & S. J. Higgins eds. (1984)]; “Animal CellCulture” [R. I. Freshney, ed. (1986)]; “Immobilized Cells And Enzymes”[IRL Press, (1986)]; B. Perbal, “A Practical Guide To Molecular Cloning”(1984). Therefore, if appearing herein, the following terms shall havethe definitions set out below.

[0066] A “DNA molecule” refers to the polymeric form ofdeoxyribonucleotides (adenine, guanine, thymine, or cytosine) in itseither single stranded form, or a double-stranded helix. This termrefers only to the primary and secondary structure of the molecule, anddoes not limit it to any particular tertiary forms. Thus, this termincludes double-stranded DNA found, inter alia, in linear DNA molecules(e.g., restriction fragments), viruses, plasmids, and chromosomes. Indiscussing the structure herein according to the normal convention ofgiving only the sequence in the 5′ to 3′ direction along thenontranscribed strand of DNA (i.e., the strand having a sequencehomologous to the mRNA).

[0067] A “vector” is a replicon, such as plasmid, phage or cosmid, towhich another DNA segment may be attached so as to bring about thereplication of the attached segment. A “replicon” is any genetic element(e.g., plasmid, chromosome, virus) that functions as an autonomous unitof DNA replication in vivo; i.e., capable of replication under its owncontrol. An “origin of replication” refers to those DNA sequences thatparticipate in DNA synthesis. An “expression control sequence” is a DNAsequence that controls and regulates the transcription and translationof another DNA sequence. A coding sequence is “operably linked” and“under the control” of transcriptional and translational controlsequences in a cell when RNA polymerase transcribes the coding sequenceinto mRNA, which is then translated into the protein encoded by thecoding sequence.

[0068] In general, expression vectors containing promoter sequenceswhich facilitate the efficient transcription and translation of theinserted DNA fragment are used in connection with the host. Theexpression vector typically contains an origin of replication,promoter(s), terminator(s), as well as specific genes which are capableof providing phenotypic selection in transformed cells. The transformedhosts can be fermented and cultured according to means known in the artto achieve optimal cell growth.

[0069] A DNA “coding sequence” is a double-stranded DNA sequence whichis transcribed and translated into a polypeptide in vivo when placedunder the control of appropriate regulatory sequences. The boundaries ofthe coding sequence are determined by a start codon at the 5′ (amino)terminus and a translation stop codon at the 3′ (carboxyl) terminus. Acoding sequence can include, but is not limited to, prokaryoticsequences, cDNA from eukaryotic mRNA, genomic DNA sequences fromeukaryotic (e.g., mammalian) DNA, and even synthetic DNA sequences. Apolyadenylation signal and transcription termination sequence willusually be located 3′ to the coding sequence. A “cDNA” is defined ascopy-DNA or complementary-DNA, and is a product of a reversetranscription reaction from an mRNA transcript. An “exon” is anexpressed sequence transcribed from the gene locus, whereas an “intron”is a non-expressed sequence that is from the gene locus.

[0070] Transcriptional and translational control sequences are DNAregulatory sequences, such as promoters, enhancers, polyadenylationsignals, terminators, and the like, that provide for the expression of acoding sequence in a host cell. A “cis-element” is a nucleotidesequence, also termed a “consensus sequence” or “motif”, that interactswith other proteins which can upregulate or downregulate expression of aspecific gene locus. A “signal sequence” can also be included with thecoding sequence. This sequence encodes a signal peptide, N-terminal tothe polypeptide, that communicates to the host cell and directs thepolypeptide to the appropriate cellular location. Signal sequences canbe found associated with a variety of proteins native to prokaryotes andeukaryotes.

[0071] A “promoter sequence” is a DNA regulatory region capable ofbinding RNA polymerase in a cell and initiating transcription of adownstream (3′ direction) coding sequence. For purposes of defining thepresent invention, the promoter sequence is bounded at its 3′ terminusby the transcription initiation site and extends upstream (5′ direction)to include the minimum number of bases or elements necessary to initiatetranscription at levels detectable above background. Within the promotersequence will be found a transcription initiation site, as well asprotein binding domains (consensus sequences) responsible for thebinding of RNA polymerase. Eukaryotic promoters often, but not always(especially in monocyte-macrophage specific promoters), contain “TATA”boxes and “CAT” boxes. Prokaryotic promoters contain Shine-Dalgarnosequences in addition to the −10 and -35 consensus sequences.

[0072] The term “oligonucleotide” is defined as a molecule comprised oftwo or more deoxyribonucleotides, preferably more than three. Its exactsize will depend upon many factors which in turn, depend upon theultimate function and use of the oligonucleotide. The term “primer” asused herein refers to an oligonucleotide, whether occurring naturally asin a purified restriction digest or produced synthetically, which iscapable of acting as a point of initiation of synthesis when placedunder conditions in which synthesis of a primer extension product, whichis complementary to a nucleic acid strand, is induced, i.e., in thepresence of nucleotides and an inducing agent such as a DNA polymeraseand at a suitable temperature and pH. The primer may be eithersingle-stranded or double-stranded and must be sufficiently long toprime the synthesis of the desired extension product in the presence ofthe inducing agent. The exact length of the primer will depend upon manyfactors, including temperature, source of primer and use the method. Forexample, for diagnostic applications, depending on the complexity of thetarget sequence, the oligonucleotide primer typically contains 15-25 ormore nucleotides, although it may contain fewer nucleotides. Theoligonucleotides herein are selected to b e “substantially”complementary to a particular target DNA or RNA sequence. This meansthat the primers must be sufficiently complementary to hybridize withtheir respective strands. Therefore, the primer sequence need notreflect the exact sequence of the template. For example, anon-complementary nucleotide fragment may be attached to the 5′ end ofthe primer, with the remainder of the primer sequence beingcomplementary to the strand. Alternatively, non-complementary bases orlonger sequences can be interspersed into the primer, provided that theprimer sequence has sufficient complementarity with the sequence orhybridize therewith and thereby form the template for the synthesis ofthe extension product.

[0073] As used herein, the terms “restriction endonucleases” and“restriction enzymes” refer to enzymes which cut double-stranded DNA ator near a specific nucleotide sequence.

[0074] A cell has been “transformed” or “transfected” with exogenous orheterologous DNA when such DNA has been introduced inside the cell. Thetransforming DNA may or may not be integrated (covalently linked) intothe genome of the cell. In prokaryotes, yeast, and mammalian cells forexample, the transforming DNA may be maintained on an episomal elementsuch as a vector or plasmid. With respect to eukaryotic cells, a stablytransformed cell is one in which the transforming DNA has becomeintegrated into a chromosome so that it is inherited by daughter cellsthrough chromosome replication. This stability is demonstrated by theability of the eukaryotic cell to establish cell lines or clonescomprised of a population of daughter cells containing the transformingDNA. A “clone” is a population of cells derived from a single cell orancestor by mitosis. A “cell line” is a clone of a primary cell that iscapable of stable growth in vitro for many generations. An organism,such as a plant or animal, that has been transformed with exogenous DNAis termed “transgenic”.

[0075] As used herein, the term “host” is meant to include not onlyprokaryotes but also eukaryotes such as yeast, plant and animal cells. Arecombinant DNA molecule or gene can be used to transform a host usingany of the techniques commonly known to those of ordinary skill in theart. One preferred embodiment is the use of vectors containing codingsequences for purposes of prokaryotic transformation. Prokaryotic hostsmay include E. coli, S. tymphimurium, Serratia marcescens and Bacillussubtilis. Eukaryotic hosts include yeasts such as Pichia pastoris,mammalian cells, insect cells and plant cells, such as Arabidopsisthaliana and Tobaccum nicotiana.

[0076] Two DNA sequences are “substantially homologous” when at leastabout 75% (preferably at least about 80%, and most preferably at leastabout 90% or 95%) of the nucleotides match over the defined length ofthe DNA sequences. Sequences that are substantially homologous can beidentified by comparing the sequences using standard software availablein sequence data banks, or in a Southern hybridization experiment under,for example, stringent conditions as defined for that particular system.Defining appropriate hybridization conditions is within the skill of theart. See, e.g., Maniatis et al., supra; DNA Cloning, Vols. I & II,supra; Nucleic Acid Hybridization, supra.

[0077] A “heterologous’ region of the DNA construct is an identifiablesegment of DNA within a larger DNA molecule that is not found inassociation with the larger molecule in nature. Thus, when theheterologous region encodes a mammalian gene, the gene will usually beflanked by DNA that does not flank the mammalian genomic DNA in thegenome of the source organism. In another example, the coding sequenceis a construct where the coding sequence itself is not found in nature(e.g., a cDNA where the genomic coding sequence contains introns, orsynthetic sequences having codons different than the native gene).Allelic variations or naturally-occurring mutational events do not giverise to a heterologous region of DNA as defined herein.

[0078] In addition, the invention also includes fragments (e.g.,antigenic fragments or enzymatically or ligand binding functionalfragments) of the apoB cell-surface receptor protein. As used herein,“fragment,” as applied to a polypeptide, will ordinarily be at least 10residues, more typically at least 20 residues, and preferably at least30 (e.g., 50) residues in length, but less than the entire, intactsequence. Fragments of the apoB cell-surface receptor protein can begenerated by methods known to those skilled in the art, e.g., byenzymatic digestion of naturally occurring or recombinant apoBcell-surface receptor protein, by recombinant DNA techniques using anexpression vector that encodes a defined fragment of apoB cell-surfacereceptor protein, or by chemical synthesis. The ability of a candidatefragment to exhibit a characteristic of apoB cell-surface receptorprotein (e.g., binding to an antibody specific for apoB cell-surfacereceptor protein, or binding to a ligand of the receptor or exhibitingpartial enzymatic or catalytic activity) can be assessed by methodsdescribed herein. Purified fragments of apoB cell-surface receptorprotein or antigenic fragments of apoB cell-surface receptor protein canbe used to generate antibodies by employing standard protocols known tothose skilled in the art.

[0079] As atherosclerotic plaques are enriched with ApoB48 expressingcells, the instant invention may also be directed toward delivery oftherapeutic agents to atherosclerotic plaques. These agents may includelabel to localize said plaques, inhibitors of apoB48R to inhibit furtherdevelopment of the plaques, or agents designed to eliminate the plaques.

[0080] A standard Northern blot assay can be used to ascertain therelative amounts of apoB cell-surface receptor protein mRNA in a cell ortissue in accordance with conventional Northern hybridization techniquesknown to those persons of ordinary skill in the art. Alternatively, astandard Southern blot assay may be used to confirm the presence and thecopy number of the gene encoding the apoB cell-surface receptor intransgenic systems, in accordance with conventional Southernhybridization techniques known to those of ordinary skill in the art.Both the Northern blot and Southern blot use a hybridization probe, e.g.radiolabeled apoB cell-surface receptor cDNA, either containing thefull-length, single stranded DNA having a sequence complementary to SEQID No. 1, SEQ ID No. 3, SEQ ID No. 7, SEQ ID No. 8, SEQ ID No. 9 or afragment of those DNA sequences at least 20 (preferably at least 30,more preferably at least 50, and most preferably at least 100consecutive nucleotides in length). The DNA hybridization probe can belabeled by any of the many different methods known to those skilled inthis art.

[0081] The labels most commonly employed for these studies areradioactive elements, enzymes, chemicals which fluoresce when exposed tospecific wavelengths of light, and others. A number of fluorescentmaterials are known and can be utilized as labels. These include, forexample, fluorescein, rhodamine, auramine, Texas Red, AMCA blue andLucifer Yellow and green fluorescent protein (GFP). The presentinvention also provides GFP-apoB48R fusion constructs that, e.g.,transfect CHO cells and confer receptor activity. A particular detectingmaterial is anti-rabbit antibody prepared in goats and conjugated withfluorescein through an isothiocyanate. Proteins can also be labeled witha radioactive element or with an enzyme. The radioactive label can bedetected by any of the currently available counting procedures. Thepreferred isotope may be selected from ³H, ¹⁴C, ³²P, ³⁵S, ³⁶Cl, ⁵¹Cr,⁵⁷Co, ⁵⁸Co, ⁵⁹Fe, ⁹⁰Y, ¹²⁵I, ¹³¹I, and ¹⁸⁶Re.

[0082] Enzyme labels are likewise useful, and can be detected by any ofthe presently utilized calorimetric, spectrophotometric,fluorospectrophotometric, amperometric or gasometric techniques. Theenzyme is conjugated to the selected particle by reaction with bridgingmolecules such as carbodiimides, diisocyanates, glutaraldehyde and thelike. Many enzymes which can be used in these procedures are known andcan be utilized. The preferred are peroxidase, β-glucuronidase,β-D-glucosidase, β-D-galactosidase, urease, glucose oxidase plusperoxidase and alkaline phosphatase. U.S. Pat. Nos. 3,654,090,3,850,752, and 4,016,043 are referred to by way of example for theirdisclosure of alternate labeling material and methods.

[0083] The following examples are given for the purpose of illustratingvarious embodiments of the invention and are not meant to limit thepresent invention in any fashion.

EXAMPLE 1

[0084] Cells and Cell Extracts

[0085] Human monocytes are isolated by adhesion to plastic fromperipheral mononuclear cells isolated on a Ficoll-Paque gradient (Boyum,1968). The adherent cells (90-95% monocytes) are used after isolation orafter 1-7 days culture in RPM1-1640 containing 20% autologous serum(Gianturco, 1994).

[0086] THP-1 monocyte-macrophages (ATCC) are grown in suspension in RPMI1640 with 10% fetal bovine serum, 2 mM glutamine, 100 μg streptomycinand 100 units of penicillin/ml and 5×10⁻⁵ M 2-mercaptoethanol. Cellswere maintained in tissue culture flasks at 37° C. in a humidifiedatmosphere of 5% CO₂ and 95% air at ≦1×10⁶ cells/ml. THP-1 monocytesdifferentiate into adherent macrophages when treated with 10⁻⁷ M phorbol12-myristate, 13-acetate (PMA) (Hara, 1987; Gianturco, 1988). Fordifferentiation, cells were seeded (1.5×10⁶ cells/35 mm³ dish) incomplete media; PMA was then added. Adherent cells were used forexperiments between 1 and 7 days. Cellular triglycerides, cholesterol,and cholesteryl ester masses (Gianturco, 1986a) were determined asdescribed. Human skin fibroblasts were early passage cells from newbornforeskin and maintained as described (Gianturco et al., 1978, 1980).

[0087] 1.5×10⁸ THP-1 monocytes were harvested and washed twice with 50ml of buffer A (0.15 M NaCl containing 50 U aprotinin/ml, 5 mMbenzamidine, and 0.1 mM PMSF) a n d resuspended in 2 ml of 20 mM Tris,pH 8.0, 50 mM NaCl, 0.1 mM EDTA, containing the protease inhibitor mixof buffer A plus leupeptin and PPACK and solubilized with 1% TritonX-114 for 15 minutes on ice. Aqueous phase extracts were prepared asdescribed (Gianturco e t al., 1988, 1994; Ramprasad et al., 1995) by themethod of Bordier (1985) and immediately frozen in liquid nitrogen afterthe addition of glycerol to a final concentration of 10% (v/v). Proteincontent was estimated by the Bradford method using the Bio-Rad ProteinAssay reagent (Bradford, 1976).

EXAMPLE 2

[0088] Purification and Microsequencing of the MBP Protein

[0089] MBP200R was purified from 75 liters of THP-1 monocytes (75×10⁹cells) by a four-step procedure, capitalizing on the unique propertiesdiscovered during other purification attempts, such as the relativeresistance of MBP200 activity to heat and to reduction. Briefly, thepurification scheme used to isolate the protein for microsequencing was:Triton X-114 aqueous-phase extracts were reduced with 2-mercaptoethanolto convert MBP200 and MBP235 to MBP200R and then heated (90-100° C.) forup to 10 minutes and centrifuged, which selectively eliminates ˜90% ofthe contaminating proteins without significant loss of MBP200R ligandbinding activity. The supernatant containing MBP200R activity wasfractionated on DEAE and then hydroxyapatite, and finally resolved bypreparative SDS-PAGE. The electroeluted MBP200R band appearedhomogeneous by two-dimensional electrophoresis and silver staining.Starting at the extraction step, this represented approximately a1200-fold purification, which reflects the difference betweenpurification from pure cells and tissues. If one can obtain sufficientcells and stabilize the desired product, cultured cells aresignificantly enriched over a tissue source since there is noextracellular material. After transfer to Immobilon, strips containingMBP200R were processed for gas-phase microsequence analysis [City ofHope Microsequencing and Mass Spectrometry Core Facility]. Twoindependent attempts indicated that the N-terminus was blocked.Therefore, the protein was trypsinized on the membrane (0.1 M ammoniumbicarbonate pH 8.0 and 10 pmole trypsin at 37° C. for 24 hours), thepeptides eluted and separated by micro-LC-chromatography on a Vydac C18column (530 μm ID) with a TFA/acetonitrile gradient; peaks were detectedby UV monitoring and analyzed by mass spectrometry and microsequencingtechniques.

[0090] Seven peptides contained sufficient mass and were sequenced; themost useful data were from peptide 29, a thirteen residue fragment inwhich 10 contiguous residues were unambiguously identified:E/L/A,A/L,Q/V/E,A,E,G,L,M,V,T,G,G,R (MH+=1333) (SEQ ID No. 5). Searchesof peptide and nucleotide sequence databases revealed this was a uniquesequence, as was peptide 18 (V/E,A/L,V,M,G,Q,M (SEQ ID No. 6)), usefulfor making oligonucleotides because of its 2 methionines. A peptidecorresponding to the unambiguous portion of peptide 29 ((C)AEGLMVTGGR;(SEQ ID No. 10)) was synthesized in the peptide core at University ofAlabama-Birmingham; the C-terminal arginine was amidated and thecysteine was added for coupling to keyhole limpet hemocyanin (KLH) andused for antibody production in rabbits. The first immunoblots of thefirst bleed were positive for all three active forms of the receptor(FIG. 4). Immunoreactivity perfectly coincided with ligand blottingactivity in the native (MBP200 and MBP235), and upon reduction, trackedthe electrophoretic mobility shift along with ligand blotting activityinto the reduced form, MBP200R; preimmune antisera did not bind to theseproteins, showing the antipeptide antibodies are specific for the MBPs.These data also confirm immunochemically the ligand blot data thatshowed that MBP235 contains MBP200 and that both are converted intoMBP200R upon reduction. Antibodies generated against GST-fusion proteinsrepresenting various domains of MBP200R verified these results.

[0091] The immunoblotting/ligand blotting experiments were repeated withnumerous THP-1 extracts and three different antipeptide antisera andaffinity-purified anti-peptide antibodies and compared to extracts ofother receptor positive and receptor-negative cells. Consistent withcell binding and ligand blotting studies, extracts of human U937monocytes, but not human skin fibroblasts or CHO extracts, showedspecific antipeptide immunoreactivity that coincided with ligandblotting activity. The antipeptide antibodies show that this uniquesequence, AEGLMVTGGR (SEQ ID No. 5), unmatched in GenBank, is indeedfrom the major ligand binding subunit, MBP200. Thus, a unique proteinthat has all of the biochemical characteristics necessary for a monocyteand macrophage-specific triglyceride-rich lipoprotein receptor waspurified.

EXAMPLE 3

[0092] cDNA Cloning and Characterization of MBP200 and MBP235

[0093] The protein/peptide microsequence data yielded two usefulpeptides from which oligonucleotides were generated. Since using highlydegenerate oligos for screening cDNA libraries is not a simple task, analternative PCR approach was sought to produce suitable probes for usein screening (Yokayama, 1993). The contiguous decapeptide sequence(obtained from a tryptic thirteen-residue fragment) was used to designnested, degenerate 17-mer oligonucleotide probes from thecarboxyl-terminal end of the peptide and from the overlappingamino-terminal end. Using a THP-1 monocyte λgt10 cDNA library (Clontech)as template DNA, the carboxy-terminal primers were used in the firstround of PCR with λ forward and reverse primers; the PCR products werefractionated over Qiagen columns to remove primers and subjected to asecond round of PCR with the more amino-terminal degenerate primers andthe λ forward and reverse primers. The second round yieldedethidium-bromide stainable products of about 150 base pairs in size. Theproducts were subcloned into a pCRII vector using a TA cloning kit(Invitrogen) and into a pBluescript II vector. Sequencing by the dideoxychain termination method indicated a clone with a 139 base pair openreading frame containing the decapeptide sequence.

[0094] Importantly, the DNA sequencing identified the first threeresidues of the peptide sequence unambiguously as Leu, Leu, Asp(Leucines were possible choices in the peptide microsequence analysis).This sequence, LLDAEGLMVTGGR (SEQ ID No. 5), correctly fits the massspectral parent ion, MH+=1333, of the LC-isolated tryptic peptides).Importantly, the DNA sequence placed an Arg immediately before the LeuN-terminal residue of the sequenced peptide, which correctly predictsthe trypsin cleavage site used to produce the peptide. Furthermore, thatthe same peptide sequence was used to produce an antipeptide antibodythat correctly and specifically identified the intact MBPs was stronglysupportive of the initial PCR results.

[0095] Based upon the PCR results, nondegenerate PCR primers weresynthesized from the original nested primers and used with the λ primersin a new PCRprotocol. PCRproducts of ˜1 kb in size were produced andsubcloned into the pCRII vector. pcr631 (SEQ ID No. 3) codes for threeof the peptides obtained by microsequence analysis of tryptic peptidefrom MBP200R and correctly predicts Arg before each. FIG. 7 shows thepredicted amino acid sequence derived from the 631 base pair PCR product(SEQ ID No. 4) and the 3 tryptic peptides are underlined.

[0096] Standard molecular biology approaches were used to clone andsequence the cDNA of MBP200 (Sambrook, 1989; Ausubel, 1987). SinceMBP200 was isolated from THP-1 monocytes, a commercial λgt10 poly dTprimed cDNA library from human THP-1 monocyte/macrophages (Clontech;Palo Alto, Calif.) was screened. The cDNA inserts range in size from0.5-4 kb, with the average size around 1 kb. The amplified librariescontain approximately 1.4×10⁶ independent clones, sufficient to includelow abundance mRNAs. The expression libraries were screened by in situplaque hybridization. The phage were plated on agarose-topped agarplates, and the plaques adsorbed onto duplicate nitrocellulose filters,denatured and probed with labeled oligonucleotides or partial cDNAs.

EXAMPLE 4

[0097] Expression of Unique MBPs in THP-1 Cells

[0098] Thus, the present studies demonstrate, as shown in FIG. 6, thatTHP-1 monocytes were surface-labeled with the affinity-purifiedanti-receptor antibody generated against a 10-residue synthetic peptidebased on an unambiguous sequence of a tryptic peptide from purifiedMBP200R. Cell-surface location of this receptor epitope confirms thatMBP200 and MBP235 are located on the cell surface, a criterion for areceptor of extracellular ligands. A unique 631 base pair PCR product(pcr631) (SEQ ID No. 3) was generated, cloned, and sequenced from aλgt10 THP-1 monocyte library using oligonucleotide primers based on thesame unambiguous tryptic peptide from MBP200R that was used to generatereceptor-specific, anti-peptide antibodies. The PCR631 product (SEQ IDNo.3) contained an open reading frame (bp3-630) which predicts a unique209-residue protein sequence (SEQ ID No. 4) that contains three of thetryptic peptides determined by microsequence analysis of purifiedMBP200R, and contains arginine residues before each of these threepeptides, correctly predicting the trypsin cleavage sites in MBP200needed to generate the microsequenced peptides (see FIG. 7). This isdirect evidence that the THP-1 λgt10 library contains MBP200-specificcDNAs. Neither the 209-residue predicted protein sequence nor thedetermined 631 nucleotide sequence has any identities or close matchesin the NCBI nonredundant peptide and nucleotide databases (PDB+SwissProt+PIR+SPUpdate+GPUpdate+GenBank+EmblUpdate+EMBL), confirming itsuniqueness. PCR631 labeled with digoxigenin was used to identifyreceptor-specific clones from the THP-1 library for sequencing.

[0099] Reverse transcriptase (RT)-PCR demonstrates that THP-1 monocytescontain mRNA coding for MBP200R. RT-PCR using primer pairs derived fromthe PCR631/MBP200R sequence and mRNA isolated from THP-1 monocytesproduced an RT-PCR product of the correct size (FIG. 8, lane 1).Notably, a second round of PCR using an internal antisense primerproduced a single, predicted 490 bp product (FIG. 8, lane 4), indicatingthe THP-1 cells from which MBP200R was purified do indeed express MBP200mRNA (FIG. 8, digitized negative image of gel photo). Restriction-sitemapping verified the sequence of the RT-PCR product, correctly producingthe predicted-size fragments for BamHI, HindIII, PstI, and XbaI (FIG. 9,digitized negative scan of gel photo). This further confirms that thesequence of PCR631 (SEQ ID No. 3), which contains coding sequences forthree tryptic peptides derived from MBP200R, and RT-PCR productsequences are correct.

[0100] The dig-labeled PCR631 probe was used to screen a THP-1 λgt10library and eight receptor clones were identified (partials, rangingfrom ˜1 to ˜3 kb in length) from ˜600,000 screened. A 3 kb clone wassequenced (λ73-3). The nucleotide sequence is unique. More 5′ probesfrom this sequence were used to screen a human placenta λgt10 randomprimed library to obtain the complete cDNA. The sequence was confirmedby sequencing multiple clones.

[0101] Reverse transcriptase experiments with PCR631-specific primersand murine P388D1 macrophages, human blood monocytes, human umbilicalvein endothelial cells, and U937 monocytes, but not human skinfibroblasts or CHO cells, produced RT-PCR products of the correct sizeand with the same restriction sites, indicating that monocytic andendothelial cells, but not fibroblasts or CHO cells, contain mRNA forthis receptor protein. These primers also produced a PCR product of thesame size and with the same restriction enzyme sites from placentalpolyA mRNA and a λgt10 human placenta library, indicating this receptormRNA is expressed in human placenta. Northern analysis revealed an mRNAof approximately 3,800 bases in RNA of human THP-1, placenta (FIG. 18),peripheral blood leukocytes, bone marrow, lymph nodes, tonsils, spleen,thymus and appendix (FIG. 19). This is in close agreement with theapproximately 3,744 bases of the full-length cDNA construct.

EXAMPLE 5

[0102] Characterization and Location of the Gene Encoding MBP200(apoB48R)

[0103] The isolation, characterization and chromosomal location of theMBP200 gene were determined in the following manner. Initially, thenumber of MBP200 genes in the human genome was determined. For this,human genomic DNA is digested with various restriction enzymes andprobed by Southern blot analyses using various portions of the MBP200cDNA. From the number of positive hybridizing bands for each digest, onecan determine whether there is a single MBP200 gene, or if MBP200belongs to a multigene family. The chromosomal location of MBP200 isprobed by in situ hybridization of metaphase chromosomes. The latteranalysis provides information on copy number if different members of thegene family are located on different chromosomes.

[0104] A human genomic library is screened by in situ plaquehybridization. Probes from both the 5′ and 3′ ends of the cDNA are usedto ensure the isolation of overlapping genomic clones. Positive clonesare isolated, and overlapping clones aligned by restriction endonucleasemapping and Southern blot hybridization. Selected regions of the geneencoding specific cDNA sequences are identified by blot hybridization,subcloned in a plasmid vector, and sequenced to identify individualgenes.

[0105] Organization of exons and introns in the gene is determined byPCR amplification of human genomic DNA with a series of gene-specificprimers, followed by cloning of the PCR products into TA vectors andsequence analysis of the inserts. Comparison of the gene sequence withthat of the cDNA would directly establish the intron-exon structure ofthe gene. There are three introns within the coding sequence. Thecharacterization of the gene from murine genomic libraries provides thebasis for analysis of MBP200 function in transgenic and knockout mice.In addition, if MBP200 is a candidate gene for subclass Pattern Bphenotype or other dyslipidemias or immune system pathologies, then theisolated MBP200 gene would be highly useful in obtaining probes for morerefined linkage analysis as well as identifying putative mutations inthe human genome. The gene is located on chromosome 16p11, as determinedby FISH.

[0106] Small, dense low density lipoprotein (subclass Pattern B) is anindependent, genetically determined coronary atherosclerosis diseaserisk factor (Krauss, 1994). Although the mechanism leading to thesubclass Pattern B phenotype is unknown, one potential scenario mayinvolve reduced clearance of chylomicrons by accessible macrophages bythis receptor and the conversion of abnormally high levels ofchylomicrons into remnants in plasma. The latter would be cleared byhepatocytes which would then down regulate hepatic LDL receptor andexacerbate the delayed remnant removal. The increased total hepaticuptake of chylomicrons remnants would increase the synthesis of apo-Bcontaining particles, and, combined with the down regulation of lowdensity lipoprotein receptor, could, in the presence of slightlyelevated triglyceride-rich lipoproteins and cholesterol ester transferproteins (CETPs), result in the conversion of normal low densitylipoproteins to small, dense low density lipoproteins, resulting in thePattern B phenotype. Since as much as 30% of chylomicrons may be takenup by bone marrow macrophages, alterations in MBP200 andtriglyceride-rich lipoprotein uptake by macrophages may be involveddirectly in the generation of the subclass Pattern B phenotype. Thus,the MBP200 (or the associated 35 kD peptide) may be candidate gene(s)for this phenotype.

EXAMPLE 6

[0107] Lipoprotein Purification

[0108] Since the receptor-binding domains appear to be in apoB-48,postprandial triglyceride-rich lipoproteins of differing flotationclasses were used to identify the domain and effects of particle size onexpression. Lipoprotein isolation was as detailed (Gianturco, 1986b)from fresh plasma containing antioxidants and protease inhibitors. Verylow density lipoprotein₁ (S_(f)100-400), very low density lipoprotein₂(S_(f)60-100), and very low density lipoprotein₃ (S_(f)20-60) wereisolated from very low density lipoprotein (d<1.006) by cumulativeflotation and sterilized as described (Lindgren, 1972; Gianturco,1986b). Chylomicrons and remnants of S_(f)>400 triglyceride-richlipoproteins were subfractionated into S_(f)>3200, S_(f)1,100-3,200, andS_(f)400-1100 by cumulative flotation through a discontinuous NaClgradient (Lindgren, 1972).

[0109] To isolate chylomicrons and remnants enriched in apoB-48, normaland hypertriglyceridemic volunteers, after a 12 hour overnight fast, eatwithin 10 minutes a meal of scrambled eggs, bread, cheese and amilkshake containing 60,000 U of aqueous vitamin A/m² body surface(Weintraub, 1987). The meal contains 50 g of fat/m² body surface, and65% of calories as fat, 20% as carbohydrate, and 15% as protein, with600 mg cholesterol/11,000 calories and a P/S ratio of 0.3. This meal was(1) well tolerated by all subjects, (2) caused a reproduciblepostprandial response, (3) gave equivalent gastric emptying rates innormal subjects and in patients, and (4) the vitamin A dose was shown tobe well within the capacity of the intestine to absorb it (Weintraub,1987). Subjects who previously volunteered in an ongoing study usingthis meal were asked to repeat the fat load and blood was isolated atpeak lipemia (as determined previously) to obtain postprandialtriglyceride-rich lipoproteins for the isolation of apoB-48-containingparticles.

[0110] Plasma chylomicron remnant fractions of S_(f)>3200 andS_(f)1100-3200 contained apoB-48 as the only apoB species (FIG. 15) andbound to the MBPs with high affinity (FIG. 16). This binding wasinhibited by anti-apoB antibodies (FIG. 17), showing that apoB-48 issufficient to mediate binding to the MBPs and apoB-100 is not necessary.Postprandial triglyceride-rich lipoproteins of different S_(f) areseparated into apoB-48 enriched particles by immunoaffinitychromatography using a monoclonal antibody that binds apoB-100 but notapoB-48 (JI-H) (Havel, 1992) and can be used directly. ApoB-100particles bind and can then be eluted (Havel, 1992). The purifiedapoB-100-only (isolated both fasting and postprandially) andapoB-48-enriched subspecies are used in cell binding and ligand blottingstudies to show that particles containing apoB-48 primarily, as well asonly apoB-100, bind to the receptor. At peak lipemia, there were atleast 3 mg protein of triglyceride-rich lipoprotein subfractions/dl infour subjects with normal fasting triglyceride levels. Immunochemicalblots demonstrated that apoB-48 comprised 30-50% of the total apoB atpeak lipemia. The apoB-48 particles were present in all subclasses, evenat 8 hrs postprandially (even in the S_(f)20-60 fraction). There were ≧6times more of each subclass in hypertriglyceridemic subjects than innormal subjects. So there are at least 900 fig of each apoB-48triglyceride-rich lipoprotein subclass from normal subjects from 100 mlof plasma at peak lipemia. Anti-CIII immunoaffinity columns usingpolyclonal antibodies provided subfractions depleted (unbound fraction)versus enriched (bound) in Apo CIII.

[0111] Protein concentrations of the lipoproteins were obtained by amodified Lowry procedure with 0.1% SDS (Lowry et al., 1951; Helenius etal., 1971). Trypsinized-VLDL, reisolated and devoid of immunochemicallydetectable apoE, was prepared as described (Gianturco et al., 1983,1986; Bradley et al., 1984). Functional loss of apoE was demonstrated bylack of binding of tryp-VLDL to partially purified bovine LDL receptorson ligand blots (Gianturco et al., 1983, 1988, 1994; Bradley et al,1984; Gianturco and Bradley, 1986). Although tryp-VLDL is devoid ofimmunochemically detectable apoE and apoCIII, it retains essentially allimmunochemically detectable apoB in fragments of 100 kDa and less asdetermined by SDS-PAGE (size) and RIA or by SDS-PAGE and quantitativedot blot analysis of parent VLDL and tryp-VLDL (Ramprasad et al., 1995).

EXAMPLE 7

[0112] Lipoprotein Modifications, Production of Model Very Low DensityLipoproteins and Lipoprotein Binding Assays

[0113] Proteolytic degradations were as described. Chemicalmodifications such as acetylation, reductive methylation, maleylation,and succinylation of lysines, cyclohexandione modification of arginineand its reversal were done (Means, 1971; Basu, 1977). Modification oflysine ε-NH₂ by acetylation or by reductive methylation had no effect onbinding to MBP200 or MBP235, but these procedures blocked binding to theLDL receptor and enhanced binding to the scavenger (acetyl LDL)receptor.

[0114] ApoB, apoE and apoCs were quantified by SDS-PAGE densitometricmethods and for integrity by Western blotting (Gianturco, 1983).Cholesterol, cholesteryl esters, triglycerides, and phospholipids werequantified enzymatically (Boehringer Mannheim kits). Total phosphoruswas determined as described (Bartlett, 1959).

[0115] Intralipids are subfractionated by cumulative flotation to removePL-rich (smaller) particles with 2.5% glycerol included to stabilize thelarge particles (Bradley, 1986). Defined lipids are used to make morephysiological very low density lipoprotein models with triglycerides,cholesteryl esters, cholesterol, and specific phospholipids (Miller andSmall, 1983). The triglyceride-rich particles are incubated with desiredapoproteins/synthetic fragments at room temperature for 2 hours andreisolated by cumulative flotation. Defined model very low densitylipoproteins are constructed with and without desired apoproteins,synthetic peptides, and fragments. Native and specificprotease-generated apoB fragments are solubilized in 1% octylglucosideto prepare model very low density lipoprotein by the dialysis method, asdone successfully with low density lipoprotein-size lipid emulsions andapoB (Ginsburg, 1984). Model studies pinpoint protein componentsinvolved in receptor binding and were used to show that apoE issufficient and apoB is not necessary for binding of largetriglyceride-rich lipoproteins to the low density lipoprotein receptor(Bradley, 1986a).

[0116] Lipoproteins were iodinated as reported (Bilheimer, 1972;Gianturco, 1986b) and samples were filtered immediately before use.Specific activities ranged from 100-200 cpm/ng protein. Less than 10% ofthe label was extracted into organic solvent. Lipoprotein bindingstudies were carried out essentially as described by Goldstein and Brown(1974). THP-1 monocytes were seeded in 6-well tissue culture plates(1.5×10⁶ cells/well) and phorbol ester (10⁻⁷ M) was added to induceadherence (Gianturco et al., 1994; Ramprasad et al., 1995). As controls,cultured human skin fibroblasts were subcultured and grown to ˜75%confluency (3-4 d after subculture at a 1:4 split ratio) in completemedium [Dulbecco's modified Eagles (DME) containing 10% NuSerum, 2 mMglutamine, 100 μg streptomycin/ml and 100 U penicillin/ml], washed withsterile saline, and preincubated in DME containing 5% LPDS for 36 h toinduce the LDL receptor (Gianturco et al., 1982). Cells were thenpreincubated for 30 min at 4° C. to cool the cells. Cells were thenincubated with RPMI-1640 (THP-1 cells) or DME (fibroblasts) containing10 mM HEPES (pH 7.4), 2 mg BSA/ml, and indicated amounts of ¹²⁵IHTG-VLDL or ¹²⁵I-tryp-VLDL alone and in the presence of 200 μg/mlunlabeled VLDL or other potential competitors for 1.5 hours at 4° C.prior to extensive washing with cold buffered saline containing 2 mgBSA/ml (Goldstein et al., 1974) as described (Gianturco et al., 1982,1983, 1988, 1994, 1986; Ramprasad et al., 1995; Bradley et al., 1984).Cells were dissolved in 0.1 N NaOH prior to the measurements ofcell-associated radioactivity and cell protein. Dishes with no cellswere used to correct for the amount of nonspecific binding to theplastic wells, as described (Gianturco et al., 1986). Duplicate dishesof cells were incubated with a range of concentrations of eachlipoprotein, alone and with excess unlabeled lipoprotein to calculatespecific binding at 4° C. (Gianturco, 1982a,b). Triton X-114solubilization of human monocytes and macrophages plasma membranes werea s described (Gianturco, 1988, 1994).

EXAMPLE 8

[0117] Antibodies and Antibody Production

[0118] Since MBP200 and MBP235 did not biochemically or immunochemicallyresemble other low density lipoprotein-like receptors or the acetyl lowdensity lipoprotein receptor (they do not cross react with antibodiesagainst the bovine low density lipoprotein receptor or the human lowdensity lipoprotein receptor-related protein), it was necessary topurify the protein. Several tissue sources were examined and the highestactivities were found in human monocytes and macrophages, which are alimited source. Thus the human THP-1 monocyte cell line was used as asource for purification, since it grows continuously in suspension witha doubling time of ˜60 hours, has identical binding proteins to humanmonocytes and macrophages, and has higher activities than the U937 humanmonocytic line.

[0119] After many attempts at ligand-affinity steps with varioustriglyceride-rich lipoprotein subfractions, both modified and native, ascaled-up two-dimensional electrophoresis procedure to obtainsufficient, partially purified MBP for monoclonal antibody productionwas used. Briefly, binding activity from plasma membrane preparationsmade from 2 billion THP-1 monocytes was solubilized with octylglucoside. Solution phase isoelectric focusing (IEF) (Rotofor apparatus)was performed. The IEF fractions containing binding activity wereconcentrated and purified by preparative SDS-PAGE and transferred tonitrocellulose or Immobilon. Binding activity was identified by ligandblotting; strips with activity were cut out for implantation in mice.Binding activity of MBP235 was lost after IEF, although MBP200 remainedactive and was used for monoclonal antibody production. Approximately2,400 hybridomas and 1,000 single cell clones were screened by twomethods: immunochemical blotting and an immunoprecipitation, followed byligand blotting, to detect loss of MBP200 binding activity. Only IgMswere generated and they had very low affinities for native MBPs.Although they immunoblotted, when coupled to Sepharose, the IgMs failedto bind MBP200 in solution. The fact that only IgMs were producedsuggested that the antigen was monocyte-macrophage specific and/or anessential, highly conserved protein.

[0120] Polyclonal antisera of high titer specific for apoB (bothcommercial and laboratory generated) were used (Bradley, 1984). Fivepolyclonal rabbit antisera to human apoE for RIA and blotting (Bradley,1984; Gianturco, 1983) were generated and used, as was a mappedmonoclonal antibody against apoB. Mab J I-H, which binds to apoB-100 butnot to apoB-48, was also used (Havel, 1992).

[0121] Sheep anti-human apoB IgG (1001400, Boehringer Mannheim,Indianapolis, Ind.) was purified by affinity chromatography using anLDL-conjugated Sepharose column prepared as described (Schneider et al.,1982). Immunoaffinity purified rabbit anti-sheep IgG conjugated toalkaline phosphatase and sheep gamma globulin was purchased from JacksonLabs (West Grove, Pa.). Rabbit anti-human apoB antibodies were isolatedb y ammonium sulfate precipitation of serum from rabbits immunizedintradermally with human LDL, isolated at d=1.03-1.05 g/ml, andemulsified in adjuvant. The anti-apoB-100 antibodies generated and/oraffinity purified were monospecific for apoB and did not recognize apoE,apoCs, or apoHDL. Anti-apoE was generated in rabbits using human apoEpurified and was monospecific for apoE. Affinity purified goatanti-apoCIII and anti-apoCII were from Dr. Ronald Krauss and Dr. G. M.Anantharamaiah, respectively.

EXAMPLE 9

[0122] Ligand Blotting

[0123] The ligand blotting assay was performed essentially as described(Gianturco et al., 1988, 1994; Ramprasad et al., 1995) with minormodifications. Aliquots of the detergent extracts were electrophoresedon 5% polyacrylamide gels containing 0.1% SDS (Laemmli, 1970) undernon-reducing conditions in a Bio-Rad minigel apparatus andelectrotransferred to nitrocellulose. After blocking for 1 hour with 5%Carnation nonfat dry milk in ligand buffer (50 mM Tris-HCl (pH 8), 90 mMNaCl and 2 mM CaCl₂), the blots were rinsed with 0.5% milk in ligandbuffer before incubation with lipoproteins in ligand buffer containing0.05% milk (Gianturco et al., 1988).

[0124] For competitive ligand blots, TGRLP were biotinylated asdescribed (O'Shannessy et al., 1984) and dialyzed extensively beforeuse. Biotin-labeled lipoproteins (with and without antisera), IgGs (the50% [NH₄]₂ SO₄ precipitate of antisera), or other potential competitorswere preincubated for 30 minutes at 4° C. and then incubated with thenitrocellulose strips for 1.5-3 hours as indicated. After extensivewashing, bound lipoprotein was detected by incubation with streptavidinlinked to alkaline phosphatase, followed by substrates BCIP and NBT(Bio-Rad Laboratories, CA). In some experiments without antibodies aspotential competitors, native, unlabeled TGRLP were used as the ligand,and bound TGRLP was detected with anti-apoB followed by alkalinephosphatase-linked secondary antibody. Ligand blots were scanned on anoptical scanner (Hewlett-Packard, Atlanta, Ga.) and binding activity wasquantitated using the Image Quant software (Molecular Dynamicsdensitometer, Sunnyvale, Calif.) as described (Gianturco et al., 1994;Ramprasad et al., 1995).

EXAMPLE 10

[0125] Membrane Binding Proteins (MBPs) for Triglyceride-RichLipoproteins

[0126] Direct binding studies at 4° C. demonstrate that humanmonocyte-macrophages, 1-6 days after isolation from blood, and humanTHP-1 monocytic cells, before and up to 7 days after differentiationwith phorbol ester, exhibit a high affinity (K_(d) 3-6 nM), saturable,specific, apolipoprotein E-independent binding site for the uptake anddegradation of certain triglyceride-rich lipoproteins. Trypsinized-verylow density lipoprotein (tryp-very low density lipoprotein) was used asthe surrogate ligand to test for the apoE-independent triglyceride-richlipoprotein binding site. This is because tryp-very low densitylipoprotein binds to the macrophage specific binding site and MBPs forhypertriglyceridemic-very low density lipoproteins (Gianturco, 1988,1994), but does not bind to low density lipoprotein receptors or torelated receptors either in cells (Gianturco, 1983; Bradley, 1984) or inligand blots (Brown, 1986) due to the absence of apoE or apoE fragmentsin tryp-very low density lipoproteins (Gianturco, 1983; Bradley, 1984).Tryp-very low density lipoproteins do not bind to other lipoproteinreceptors, such as those in the scavenger receptor pathway (Gianturco etal., 1988, 1994). Notably, tryp-very low density lipoproteins retainessentially all of the parent very low density lipoproteins'immunochemically detectable apoB, in fragments of ˜100 kDa and less. Useof ligands with mutually exclusive receptor specificities simplifies theinterpretation of comparative and competitive binding studies b yreducing the ambiguity seen when hypertriglyceridemic-very low densitylipoprotein is used, since hypertriglyceridemic-very low densitylipoprotein also binds to the low density lipoprotein receptor via apoE(Gianturco, 1983). Moreover, the absence of apoE in tryp-very lowdensity lipoprotein would reduce the potential binding to other membersof the low density lipoprotein receptor gene family that bind via apoE,such as low density lipoprotein receptor-related protein (Beisiegel,1989; Kowal, 1989; Kowal, 1990) and the related very low densitylipoprotein receptor (Takahashi, 1992). Studies were conducted at 4° C.in prechilled, washed cells to preclude secretion of apoE or otherconfounders, such as lipoprotein lipase, which modulate/mediatelipoprotein binding to the low density lipoprotein receptor familymembers. In addition, the medium contained no serum components to avoidadding any apoE that could be in lipoprotein-deficient serum (LPDS).

[0127] Ligand blots identified two membrane binding proteins of apparentMW of approximately 200 and approximately 235 kDa (MBP200 and MBP235) inboth cell types. The MBPs share the same ligand specificity as thecellular site and bind dietary plasma chylomicrons S_(f)>400 from normalsubjects, hypertriglyceridemic very low density lipoprotein, and thesurrogate ligand trypsinized very low density lipoprotein devoid of apoE(tryp-very low density lipoprotein). The MBPs also bind t apoE-freeB-VLDL from apoE knockout mice; this VLDL contains apoB48 as thepredominant apoB species. The MBPs do not bind with high affinity lowdensity lipoproteins, acetyl low density lipoprotein or normal very lowdensity lipoprotein. Neither lipoprotein lipase nor apoE are requiredfor triglyceride-rich lipoprotein binding to the cells or the isolatedMBPs. The cellular binding site and the MBPs are expressed a t similarlevels at all stages of differentiation, unlike the low densitylipoprotein or the acetyl low density lipoprotein receptor.Triglyceride-rich lipoproteins, which bind to the MBPs, induce rapid,saturable cellular triglyceride accumulation in monocytes as well a smacrophages; normal very low density lipoprotein does not. In addition,the cell binding site and MBP200 and MBP235 are not affected by themedia sterol content, unlike the low density lipoprotein receptor. Takentogether, these data indicate that human monocytes and macrophagesexhibit a high affinity, saturable, specific, apoE- and lipoproteinlipase-independent binding site and MBPs for triglyceride-richlipoproteins which differ in expression, specificity, and molecular sizefrom receptors of the low density lipoprotein receptor gene family orthe acetyl low density lipoprotein receptor (Gianturco et al., 1994).The characteristics of MBP200 and MBP235 suggest that they arecandidates for the receptor-mediated, apoE-independent uptake ofhypertriglyceridemic-very low density lipoprotein and chylomicrons bymonocytes and macrophages. Therefore, MBP200 and/or MBP235 may beinvolved in cellular nutrition or, when overwhelmed, in foam cellformation.

EXAMPLE 11

[0128] Triglyceride-Rich Lipoprotein Receptors with Unique Properties

[0129] Protease and heparinase susceptibility studies demonstrate that(1) these MBP activities have essential protein components, but notheparan sulfate proteoglycan components; (2) the MBPs are located on thecell surface; (3) heparan sulfate proteoglycans do not facilitatetriglyceride-rich lipoprotein binding to this specific cellular site(Ramprasad et al., 1995).

[0130] To determine protease effects on the high affinity,apoE-independent cell binding sites, THP-1 macrophages were treated withRPMI-1640 alone (control) or with 3 μg pronase/ml at 37° C. for 40minutes prior to the binding studies at 4° C. These conditions have beenshown to have no effect on cell growth, but to alter cell surfacereceptors (Burger, 1970; Goldstein & Brown, 1974). The cell and ligandconditions used precluded potential ambiguities introduced b y alternatelipoprotein pathways. Pronase pretreatment of cells significantlyreduced the binding of ¹²⁵I-tryp-very low density lipoprotein by 50-65%compared to controls (viability>95%). Recovery of the binding oftriglyceride-rich lipoprotein to cells (>80%) occurred within 2 to 4hours after the pronase-treated cells were washed and further incubatedat 37° C. in complete medium prior to assessment of binding at 4° C.Ligand blots demonstrated that THP-1 monocytes treated with 3 μgpronase/ml for 40 minutes a t 37° C. lost approximately 50-60% of theirMBP200 and MBP235 activities, as compared to control cells. After the 4hour recovery period in complete media, total MBP activities in controla n d pronase-treated cells were equal and somewhat greater than seenimmediately after treatment, in parallel with the kinetics of recoveryof triglyceride-rich lipoprotein binding to cells. Thus, proteolysisexperiments establish the cell surface location of both MBPs. Theparallel pronase-induced loss and subsequent recovery of MBP bindingactivity and cellular binding of triglyceride-rich lipoprotein stronglysupport their role as triglyceride-rich lipoprotein receptors.

[0131] Protease susceptibility did not rule out the possibility that theactivities were proteoglycans. Moreover, others have reported thatproteoglycans, specifically heparan sulfate proteoglycan, augmentedand/or facilitated lipoprotein binding to the low density lipoproteinreceptor (Mulder 1992), to low density lipoprotein receptor-relatedprotein (Nykjaer, 1993), and to the very low density lipoproteinreceptor (Takahashi, 1994). Heparinase treatment of THP-1 extracts hadno effect on either MBP activity in ligand blots, indicating these MBPsare not themselves heparan sulfate proteoglycans. Likewise, there was nodecrease in the total, nonspecific, or specific binding ofhypertriglyceridemic-very low density lipoprotein or tryp-very lowdensity lipoprotein to THP-1 macrophages pretreated with activeheparinase prior to 4° C. binding studies. The heparinase experimentsdemonstrated that neither MBP200 nor MBP235 nor the equivalent cellulartriglyceride-rich lipoprotein binding site were heparan sulfateproteoglycan.

EXAMPLE 12

[0132] Effects of Reduction on MBP200 and MBP235

[0133] The LDL receptor, LDL receptor-related protein and the VLDLreceptor have multiple cysteine-rich domains which comprise theircomplex ligand binding domains and specificities. The low densitylipoprotein receptor loses activity upon reduction (Daniel, 1983) andthe other lipoprotein receptors would be expected to lose bindingactivity upon reduction. For example, the scavenger receptor's trimericstructure (MW˜260 kDa) could be selectively reduced with thiols toactive monomers, but upon exhaustive reduction, all ligand bindingactivity was lost (Via, 1992). In contrast, reduction of MBP200 andMBP235 generated a new active species, MBP200R, which retained fullactivity even under exhaustive reduction conditions (boiling in 100 mMDTT or 5% 2-mercaptoethanol (2-ME)). In addition, although disulfideswere present in both MBPs (both mobilities changed upon reduction),these disulfides were not essential for ligand binding activities.

[0134] To demonstrate that MBP200R arose from both MBP200 and MBP235,two-dimensional (2-D) SDS-PAGE was used to separate the MBPs on thebasis of size, both before and after reduction with 2-ME. In manyindependent experiments, both MBP200 and MBP235, in the absence ofreduction in the second dimension, retained their original, distinctmobilities and activities (FIG. 1A, seen on the diagonal). In contrast,upon reduction in the second dimension, both MBP200 and MBP235mobilities were altered relative to the ˜200 kDa standard, and migratedwith identical mobilities (seen off the diagonal), intermediate betweenMBP200 and MBP235, (FIG. 1, panel B) (Ramprasad et al., 1995). Thisdemonstrates unequivocally that MBP200R arises from both MBP200 andMBP235. These changes in electrophoretic mobility reflect reduction ofintramolecular disulfide bonds and, in the case of MBP235 where itselectrophoretic mobility increased upon reduction, the possible loss ofa small subunit(s) not involved in ligand binding. These conclusionswere confirmed with receptor-specific antibodies.

EXAMPLE 13

[0135] Ligand Binding Affinities of MBP200, MBP235 and MBP200R

[0136] The 2-D experiments proved that both MBP235 and MBP200 areconverted into MBP200R upon reduction. The ligand binding properties ofeach MBP was measured before and after reduction by quantitative ligandblotting analyses detected with anti-apoB antibodies and quantified byscanning densitometry (Ramprasad et al., 1995). MBP200 and MBP235exhibited similar, high affinity saturable binding, with saturationoccurring between 5 and 10 μg apo very low density lipoprotein/ml (FIG.2A&B). Scatchard analyses of the data indicate that MBP200 and MBP235have similar K_(d)s of 1.6 and 2.2 μg apo very low densitylipoprotein/ml and B_(max) of 96 and 66 ng apo very low densitylipoprotein/mg cell protein, respectively (FIG. 2C).

[0137] The binding of hypertriglyceridemic-very low density lipoproteinto MBP200R exhibits similar, high affinity, saturable binding with aK_(d) of 1.4 μg/ml and a B_(max) of 160, approximately equal to the sumof the maximal binding activities of its precursors, MBP200 and MBP235(FIG. 2C). Indeed, the theoretical curve (dashed line) obtained byadding the amount of very low density lipoprotein bound to MBP200 andMBP235 at each level of lipoprotein is nearly superimposable on themeasured binding curve for MBP200R (FIG. 2B). The binding affinities ofhypertriglyceridemic-very low density lipoprotein and tryp-very lowdensity lipoprotein for the MBPs were similar to their bindingaffinities for intact human blood-borne and THP-1 monocytes andmacrophages, where K_(d) of 2-4 μg apo very low density lipoprotein/mlwere determined (Gianturco, 1994). This striking similarity in ligandaffinities provides additional supporting evidence that these MBPs areresponsible for the apoE-independent, high affinity and saturablebinding of triglyceride-rich lipoproteins to monocytes and macrophages.

EXAMPLE 14

[0138] Thermal Conversion of MBP235 into MBP200 Binding Activity

[0139] The reduction data indicated that MBP200 and MBP235 share acommon protein component of approximately 200 kDa, which retains all theligand binding activity. Since the mobility of MBP235 increased uponreduction, this suggested that one or more small subunits may be presentin MBP235 and lost, either due to reduction of an intermoleculardisulfide bridge(s) or by an allosteric process caused by the disruptionof the intramolecular disulfides during reduction of the 200 kDaprotein. At 65° C., MBP235 was converted into MBP200 without loss oftotal binding activity, suggesting heat dissociates a small subunit(s)not required for ligand binding from a common large protein subunit thatbinds triglyceride-rich lipoproteins. Thus, MBP235 can be converted intoMBP200 with full retention of ligand binding activity. Moreover, thesedata suggest that MBP235 has (a) noncovalent subunit(s) of approximately35 kDa total mass that is/are lost upon heating or disruption of theintramolecular cysteines of the parent backbone shared by MBP235 andMBP200. Additionally, the subunit(s) is/are not involved directly intriglyceride-rich lipoprotein binding. The subunits are not the receptorassociated protein that interats with and modulates members of the LDLreceptor family, as determined immunochemically.

[0140] In a working model (FIG. 3), MBP235 is comprised of two subunits,MBP200 and a ˜35 kDa subunit, consistent with its apparent molecularweight on SDS-PAGE. The MBP235 complex dissociates upon heating into theactive MBP200 subunit and an inactive smaller subunit(s). Uponreduction, the MBP235 complex also dissociates, losing its smallersubunit, and its active MBP200 component is extended by the reduction ofintramolecular cysteines, yielding an active binding protein (MBP200R)with a slightly slower migration, intermediate between MBP235 and MBP200mobilities. MBP200 lacks the associated subunit(s), has saturationbinding characteristics like those of MBP235, and upon reduction, isextended due to loss of its intramolecular disulfide bonds, yielding aproduct with identical electrophoretic mobility as that produced byreduction of the MBP235 complex. The model thus emphasizes theconclusion that a large and similar, if not identical, protein backboneis common to both MBP200 and MBP235 and contains the ligand bindingdomain(s). This suggests that the ligand binding domain(s) are notlocated either in domain(s) involved in the structural changes thatoccur upon reduction, or in domains that bind to the putative 35 kDasubunit. The conclusions reached from the reduction andheat-dissociation studies based on ligand blotting analyses have beenconfirmed using the antipeptide antibodies derived from MBP200.

EXAMPLE 15

[0141] Expression of MBP200 Confers Receptor Activity onReceptor-Negative Cells

[0142] The reconstruction and sequence characterization of the MBP200cDNA has been completed to determine the primary structure of thisunique protein and to express the protein in receptor-negative cells(CHO, fibroblasts, COS). This process examines (1) if MBP200 issufficient for receptor function; (2) if MBP200 possesses functionalligand binding domains; and (3) whether the effects of receptorexpression on the cellular binding and uptake of triglyceride-richlipoproteins is independent of macrophage-associated apoE and/orlipoprotein lipase. Eight cDNA clones were identified in the THP-1library and four in the placenta library. The MBP cDNA was reconstructedfrom overlapping cDNAs by the direct ligation of appropriate restrictionfragments. Isolation of the full-length cDNA was verified by comparingits size (3744 bases) to the size of the mRNA (about 3.8 kb), andverified by the complete sequencing of the cDNA.

[0143] The complete characterization of the cDNA provides informationnecessary for expressing the protein in vitro and for thecharacterization of the corresponding gene. Characterization of the cDNAalso provides information on the primary structure of the protein, thepotential functional binding domain of the protein and possibly therelationship of the 35 kD subunit(s) to MBP200, as it is possible thatthe subunit is derived by post-translational proteolysis as has beenshown for the low density lipoprotein receptor-related protein (˜600kDa=520+85 kDa).

[0144] Obtaining a full-length MBP200 cDNA allows the functions of theprotein in vitro in receptor-negative cells to be shown in detail. TheMBP200 cDNA was cloned into a pCDNA vector downstream of thecytomegalovirus promoter and transfected with a selectable marker gene(neomycin) into receptor-negative cells (CHO). Stable and transienttransformants are then selected with G418, and mass cultures andspecific clonal lines examined for receptor activity by incubation withDiI-labeled tryp-very low density lipoprotein. Uptake of the fluorescentlabel by cells transfected with pCDNA plus receptor cDNA, but not bycells transfected with the pCDNA vector alone, demonstrates that thecDNA is sufficient to confer full receptor activity, and hence, theplasma membrane targeting of the receptor. The number of gene copiesincorporated into stable transformants can be determined and MBPexpression evaluated. Using both cDNA and antibody probes, theexpression of both the mRNA and proteins, as well as the membranetargeting of MBP200, is determined. By comparing the size of MBPexpressed in vivo to the protein synthesized by cell translation invitro, one can obtain information on the intracellular processing ofMBP200 and determine if the 35 kD protein is proteolytically derivedfrom MBP200. For the latter analysis, messenger RNA corresponding toMBP200 is synthesized by run-off transcription using either T7 or T3 RNApolymerase in the presence of pGppG.

[0145] As receptor function is evident from expression of the DNAencoding MBP200, characterization of the protein domains involved inligand binding can also be determined. 5′-deletion and site-directedmutants are constructed and expressed in stably transformedreceptor-negative cells. By examining plasma membrane targeting andstability of the protein using the antibodies against MBP200, one canidentify domains of the proteins important for ligand binding.

[0146] In addition, as receptor function is mediated by MBP200 alone,increased expression of MBP200 should increase triglyceride-richlipoprotein uptake and downregulation should diminish uptake. MBP200 isplaced under tetracycline (tet) regulation using a minimal CMV promoterfused to tet operator (0) sequences (tetO-CMV) in cells expressing ahybrid, tet-controlled transactivator (tetTA, the tet repressor DNAbinding domain fused to the transactivation domain of VP16 from HSV thatis essential for transcription). TetTA stimulates tetO-CMV promoters inthe absence of tet (Gossen and Bujard, 1992). Tet binds to tetTA withhigh affinity and prevents its binding to the tetO-CMV promoter, therebysilencing the promoter. Varying the concentration of tet allowsdown-regulation of gene expression by up to 5 orders of magnitude. Thus,using such a system, one can examine the effect of expression of MBP ontriglyceride-rich lipoprotein uptake and determine if there is a directdose-dependent effect on uptake. To obtain receptor-negative cell linesexpressing the tetTA, stable transformants containing plasmids encodingtetTA and the hygromycin-resistance gene are isolated, and linesexpressing tetTA and capable of regulating a transfected reporter genedriven b y tetO-CMV selected. These are also now commercially available.The receptor-negative, tetTA-expressing cell lines are then transfectedwith MBP200 driven by the tetO-CMV promoter with a neomycin resistancegene, and neomycin-, hygromycin-resistant cells selected. Transformantsare examined for the expression of MBP200, ligand binding andtriglyceride-rich lipoprotein uptake in the absence (full expression)and presence of varying tet concentrations to control the degree ofdownregulation. Alternatively, other regulatable promoters (e.g.metallothionein) can be used.

EXAMPLE 16

[0147] The Smaller MBP200-Associated Subunit(s)

[0148] The ˜35 kD protein(s) associated with MBP200 does not appear tobe the receptor-associated protein described (Strickland, 1990). Threelines of evidence for this conclusion include: (1) rabbit anti-humanreceptor-associated protein antibodies did not exhibit cross-reactivitywith MBP235 in immunoblots, while readily detecting purified humanreceptor-associated protein and free receptor-associated protein inextracts of human skin fibroblasts and THP-1s; (2) receptor-associatedprotein did not associate with MBPs following ligand blotting underconditions where receptor-associated protein binds to members of the lowdensity lipoprotein receptor gene family; and (3) receptor-associatedprotein did not compete for hypertriglyceridemic-very low densitylipoprotein binding to MBP200 or MBP235, even when in 1000-fold molarexcess, but readily competed for the binding of this ligand to the lowdensity lipoprotein receptor. It is highly likely, therefore, that the35 kD protein(s) corresponds to a unique MBP-associated protein.

EXAMPLE 17

[0149] Ligand Identification

[0150] Neither apoE nor lipoprotein lipase is required for binding oftriglyceride-rich lipoprotein to monocytes and macrophages or to MBP200and MBP235 on ligand blots (Gianturco, 1994). Anti-apoB antibodiesinhibit binding of hypertriglyceridemic-very low density lipoprotein andtryp-very low density lipoprotein, devoid of apoE, to both THP-1 cells(FIG. 12) and MBP200 and MBP235 on ligand blots (FIGS. 5, 10 and 11).Control, nonimmune IgG tested at the same level had no effect on bindingof triglyceride-rich lipoprotein to either cells (FIG. 12) or MBP200 orMBP235 on ligand blots (FIGS. 5, 10 and 11). The region of apoB thatbinds to the receptor is in the apoB-48 region (the N-terminal 48% ofapoB-100), since chylomicrons containing apoB-48, but not apoB-100, bindwith high affinity (Gianturco, 1995) (FIG. 16), and this binding isinhibited specifically by antibodies against apoB (FIG. 17).

[0151] Competitive 4° C. cell binding and ligand blotting studiesdemonstrated that pretreatment and/or coincubation of cells (Table 1)and blots (FIG. 13) with heparin (up to 10 mg/ml) or lactoferrin (up to500 μg/ml) had no effect on the apoE-independent binding oftriglyceride-rich lipoprotein to cells or to MBP200 and MBP235, or onthe triglyceride accumulation induced by triglyceride-rich lipoproteinin THP-1 cells. Since heparin pretreatment removes surface-boundlipoprotein lipase, and coincubation causes release of surface-boundlipoprotein lipase thereby enhancing triglyceride accumulation inducedby triglyceride-rich lipoprotein when lipoprotein lipase is on the cellsurface, these studies demonstrate that lipoprotein lipase is notnecessary for the observed interactions. In contrast to studies with lowdensity lipoprotein receptor family members, lipoprotein lipase at 1-2mg/ml showed partial inhibition (-25% of specific binding) duringco-incubations (FIG. 14; Table 1), and had no effect when blots werepretreated with lipoprotein lipase and then incubated withtriglyceride-rich lipoprotein. Thus, lipoprotein lipase does notinteract with the MBPs directly, but the partial inhibition duringcoincubation suggests that the binding domain for the MBPs in apoB isnear the lipoprotein lipase binding site(s) of apoB, which has beenshown to be in the N-terminal region. Thus, the receptor binding domainsare in the N-terminal apoB region at or near the lipoprotein lipasebinding site and not in a heparin binding domain, which is consistentwith the observed binding of apoB48-containing chylomicrons to cells andto MBP200 and MBP235.

[0152] Thus, the binding determinant(s) appear to be in apoB. All nativetriglyceride-rich lipoproteins were isolated in the presence ofantioxidants and show no evidence of oxidation, such as thiobarbituricacid reacting species (TBARs) or apoB fragmentation that oxidationproduces. Thus, oxidation of triglyceride-rich lipoproteins is notrequired for their binding to this receptor. Although oxidized lowdensity lipoprotein can compete for binding to the cellular site, itbinds to numerous monocyte and macrophage proteins, and the competitionappears to be non-specific.

EXAMPLE 18

[0153] Anti-apoB Antibodies Inhibit the Binding of HTG-VLDL S_(f)100-400to MBP200 and MBP235

[0154] Previous experiments in human blood-borne and THP-1 monocytes andmacrophages (Gianturco et al., 1994; Ramprasad et al., 1995) with thesurrogate ligand, trypsinized-VLDL, which retained essentially all apoBimmunoreactivity (in fragments of ≦100 kDa) but was devoid ofimmunochemically detectable apoE (Gianturco et al., 1983, 1986; Bradleyet al., 1984) and apoCIII and failed to bind to the LDL receptor incells (Gianturco et al., 1983) and in ligand blots (Gianturco et al.,1988), suggested that apoB may be the ligand for thismonocyte-macrophage cell site and corresponding MBPs. Thus competitiveligand blotting experiments were done with several polyclonal anti-apoBspecific antibodies to determine if these were capable of specificallyblocking binding of HTG-VLDL to the putative TGRLP receptor proteinsMBP200 and MBP235.

[0155] In the representative experiment shown in FIG. 10, THP-1 monocyteextracts were electrophoresed and transferred to nitrocellulose,blocked, and incubated with biotinylated VLDL in the absence (lane 1)and the presence of a monospecific rabbit (1325) anti-apoB IgG (40μg/ml, lane 2; 400 μg/ml, lane 3), or the corresponding preimmune IgG(rabbit 1325) (90 μg/ml, lane 4; 400 μg/ml, lane 5). Binding ofbiotinylated HTG-VLDL S_(f)100-400 was visualized withstreptavidin-linked alkaline phosphatase and the image digitized (FIG.10A) and quantified by scanning densitometry (FIG. 10B). The lower levelof anti-apoB IgG (40 μg/ml, lane 2) blocked ˜50% of binding, and thehigher level (400 μg/ml, lane 3) blocked all visibly detectable HTG-VLDLbinding to MBP200 and MBP235 (˜80% by densitometry). In contrast, thepreimmune IgG at 90 μg/ml blocked none of the binding (lane 4), and at400 μg/ml, blocked 25% (lane 5). In a separate ligand blottingexperiment, this apoB antibody did not inhibit the binding of the samebiotinylated VLDL to the bovine LDL receptor. FIG. 10 is representativeof 12 separate experiments with four different anti-apoB antibodies; inall cases, anti-apoB antibodies specifically inhibited binding ofHTG-VLDL, chylomicrons, or tryp-VLDL to MBP200 and MBP235. Thus, apoB isinvolved in the binding of HTG-VLDL to the MBPs. These results areconsistent with studies demonstrating specific, high affinity binding oftryp-VLDL and implicate apoB in the binding of TGRLP to MBP200 andMBP235. Alternatively, the anti-apoB antibodies could sterically hinderanother component of the ligand for MBP200 or MBP235.

EXAMPLE 19

[0156] Antibodies Against Other Apoproteins of HTG-VLDL Fail to Inhibitits Binding to MBP200 or MBP235

[0157] ApoB is only approximately 30% of the total protein mass inHTG-VLDL S_(f)100-400; apoE is 6-8%, and apoCs are ˜63% (Gianturco etal., 1980, 1983). On a molar basis, HTG-VLDL SflOO-400 contains 1 moleof apoB, approximately 3-6 moles of apoE, and >150 moles apoCs(primarily apoCIII) per mole VLDL. To directly determine if any of theseother apoproteins are the ligand sterically hindered by the anti-apoBantibodies in the experiments represented by FIG. 10, or if theseapoproteins contribute to the binding of HTG-VLDL to MBP200 and MBP235,a series of competitive ligand blotting experiments with polyclonalantibodies against the other major apoproteins of HTG-VLDL wereperformed. All antibodies recognized their antigens in native VLDL andthe anti-apoCIII antibody was used to isolate apoCIII-rich VLDLs.

[0158] In FIG. 11, MBP200 and MBP235 activities (either or both) appearas a complex of two or more bands due to the existence of severalpermissible oxidation states and/or disulfide isomers, as previouslypublished (Ramprasad et al., 1995). In the experiment shown in FIG. 11A,biotinylated HTG-VLDL S_(f)100-400 incubated with buffer (lane 1) orwith nonimmune IgG (lane 3) binds to MBP200 and MBP235 to a similarextent. Neither anti-apoE (lane 4) nor anti-apoCIII (lane 5) diminishesthe binding of HTG-VLDL to MBP200 or MBP235, but anti-apoB againeffectively blocks >90% of the binding of HTG-VLDL (lane 2 and FIG.11B). That anti-CIII antibody failed to block binding of HTG-VLDL toMBP200 or MBP235, even though the total apoCIII mass is approximatelytwo times the mass of apoB in HTG-VLDL S_(f)100-400, argues against thealternative explanation offered above that the anti-apoB antibodiesblock by sterically hindering another apoproteins interaction with theMBPs. In other ligand blotting experiments, the concentration ofanti-apoE IgG used in these experiments blocked all binding of HTG-VLDLto the LDL receptor. Competitive ligand blotting studies withanti-apoCII IgGs demonstrated that these antibodies do not inhibitbinding. Taken together, the competitive ligand blotting studiesstrongly suggest that apoB, but not apoE, apoCIII, or apoCII, mediatesthe binding of HTG-VLDL to MBP200 and MBP235.

EXAMPLE 20

[0159] Anti-apoB Antibodies Inhibit the Binding of TGRLP to the TGRLPReceptor of THP-1 Monocytes but not to the LDL Receptor of Fibroblasts

[0160] To confirm that apoB mediates the binding of TGRLP to thelipoprotein lipase- and apoE-independent TGRLP cellular receptor,competitive cell binding studies with THP-1 monocyte-macrophages wereconducted under experimental conditions which minimize the expression ofthe LDL receptor, the LDL receptor-related protein/α₂ macroglobulinreceptor (LRP), lipoprotein lipase, and apoE (one day after adherencewas induced by PMA) as described (Gianturco et al., 1994). As a control,competitive binding studies were also done simultaneously with culturedhuman skin fibroblasts with upregulated LDL receptors, since HTG-VLDLS_(f)100-400 binds to the LDL receptor via apoE and not via apoB(Gianturco et al., 1983; Bradley et al., 1984). Consistent with theligand blotting studies (FIG. 10), the high affinity, specific bindingof ¹²⁵I-HTG-VLDL to THP-1s was inhibited by antibodies to apoB but notby the equivalent level of nonimmune IgGs (FIG. 12A). In contrast, andindicating the specificity of the blocking experiments in THP-1s, thesame anti-apoB antibody did not inhibit the LDL receptor specificbinding of ¹²⁵I-HTG-VLDL to the fibroblasts (FIG. 12B), consistent withprevious studies. This representative experiment shows that theinhibition of ¹²⁵I-HTG-VLDL binding to THP-1s by anti-apoB antibodieswas not significantly different from the inhibition by homologous,unlabeled HTG-VLDL. This indicates that apoB is the component of TGRLPresponsible for their high affinity, specific binding to THP-1 cellswhen the LDL receptor, LRP, lipoprotein lipase, and apoE are suppressed.

EXAMPLE 21

[0161] Effects of Lactoferrin, Heparin, and Lipoprotein Lipase onBinding of HTG-VLDL S_(f)100-400 to MBP200 and MBP235 and to THP-1Monocyte-Macrophages

[0162] A series of competitive ligand blotting studies were carried outto further distinguish MBP 200 and MBP235 from receptors of the LDLreceptor family and to further delineate the binding domains in apoB forthis distinct receptor. As shown in FIG. 13, neither lactoferrin norheparin is effective inhibitors of the binding of HTG-VLDL to MBP200 andMPB235. Ir this representative experiment, nitrocellulose stripscontaining MBP200 or MBP235 were incubated with 0.5 μg of biotinylatedHTG-VLDL/ml in the absence (lane 1) or in the presence of lactoferrin at50 μg protein/ml (lane 2) or 500 μg protein/ml (lane 3); heparin at 10U/ml (lane 4) and 100 U/ml (lane 5); or HTG-VLDL at 25 μg/ml (lane 6) or5 μg/ml (lane 7). Binding of biotinylated VLDL was visualized withstreptavidin-linked alkaline phosphatase (FIG. 13A) and quantified byscanning densitometry (FIG. 13B). Only unlabeled HTG-VLDL effectivelycompeted with biotinylated HTG-VLDL for binding to MBP200 and MBP235.Lack of inhibition of TGRLP binding to MBP200 and MBP235 by heparin atlevels that are known to displace lipoprotein lipase from cellsindicates that binding is not mediated by lipoprotein lipase potentiallybound to the VLDL. It also suggests that the apoB domain involved inbinding to MBP200 and MBP235 is different from the apoB domain involvedin the binding of LDL to the LDL receptor, since the apoB-LDL receptorinteraction is disrupted by heparin (Goldstein et al., 1974). Further,it suggests that the domain in apoB that binds to MBP200 and MBP235 isnot in a heparin-binding domain. Lack of inhibition of binding bylactoferrin indicates that MBP200 and MBP235 are distinct from theputative hepatic remnant receptors (van Berkel et al., 1995).

[0163] To further characterize the receptor binding domain in apoB ofHTG-VLDL, and distinguish its binding to the monocyte TGRLP receptorfrom binding to the LDL receptor or related receptors, competitiveligand binding studies were carried out with levels of lipoproteinlipase reported to enhance binding of lipoproteins to LDL receptorfamily members or to heparan sulfate proteoglycans (HSPG) on cells. Inthe representative experiment shown in FIG. 14, THP-1 monocyte extractswere electrophoresed and transferred to nitrocellulose. BiotinylatedHTG-VLDL (3 μg/ml) was preincubated for 30 min at 4° C. (to inhibitpotential lipolysis by lipoprotein lipase) and then with thenitrocellulose strips for 3 h at 4° C. with buffer (lane 1) or withlipoprotein lipase (at 0.2, 2.0, 20 μg/ml; lanes 2-4) or with the samelevels of bovine serum albumin as controls (lanes 5-7). Surprisingly,lipoprotein lipase, at levels which enhance binding to LRP, blocks thebinding of HTG-VLDL to MBP200 and MBP235 in a concentration-dependentmanner (FIG. 14B). In contrast, albumin has minimal effects on HTG-VLDLbinding. Thus, lipoprotein lipase does not mediate the interaction ofVLDL with this monocyte-macrophage receptor, rather, it inhibitsbinding. Inhibition of binding by LpL is likely due to its binding tothe N-terminal domain of apoB (Choi et al., 1995), since preincubationof the receptors on nitrocellulose strips with lipoprotein lipase failedto inhibit the subsequent binding of TGRLP.

[0164] Competitive cell binding studies also demonstrated thatlactoferrin fails to significantly inhibit the binding of tryp-VLDL orHTG-VLDL to monocyte-macrophages (<5%), and high levels of heparin (10mg/ml) have only a small effect (≦18%) (Table 1). Other studies withheparin at lower levels (1 mg/ml) show little to no inhibition of TGRLPbinding. These binding studies also indicate that lipoprotein lipase, atlevels shown by others to enhance binding of lipoproteins to cellularHSPG (Eisenberg et al., 1992; Rumsey et al., 1992), to LRP (Beisiegel etal., 1991; Chappel et al., 1993) and to the LDL receptor (Mulder et al.,1993) (1-2 μg/ml), do not enhance uptake of tryp-VLDL, rather theypartially inhibit binding (to 26% a t 1.6 μg lipoprotein lipase/ml).That the inhibition of binding of TGRLP to cells by lipoprotein lipaseis less than the inhibition of TGRLP binding to MBP200 or MBP235 islikely due to the competing enhancement of lipoprotein binding tocellular HSPGs by lipoprotein lipase, which is not a confounder inligand blots. Thus, the results of cell binding studies are similar tothe results of ligand blotting studies, with inhibition of binding byanti-apoB and by lipoprotein lipase, but not by pre- or non-immune IgG,lactoferrin, or heparin. TABLE 1 Competition of specific TGRLP bindingto THP-1 monocyte-macrophages Percent Inhibition Experiment 1 Experiment2 Experiment 3 Additions ¹²⁵I-tryp-VLDL₁ ¹²⁵I-HTG-VLDL₁ ¹²⁵I-tryp-VLDL₂Buffer (control)  0  0  0 Lactoferrin  4*  5* ND LpL  26^(†) ND  17^(†)Heparin  14  18 ND (10 mg/ml) Unlabeled 100 100 100 VLDL (20x) #inhibition, with unlabeled VLDL inhibition as 100%. The specific binding(0% inhibition) ranged from ˜140 to ˜20 fmol/mg cell protein reflectingthe particles differing affinities (tryp-VLDL₁ > HTG-VLDL₁ >tryp-VLDL₂).

EXAMPLE 22

[0165] Chylomicrons S_(f)>1100 Containing apoB-48 but not apoB-100 Bindto MBP200 and MBP235

[0166] The specific inhibition of HTG-VLDL and tryp-VLDL binding tocells and MBP200 and MBP235 on ligand blots by antibodies to apoBindicate that this apoprotein is necessary for the binding of HTG-VLDLto this receptor. The inhibition by LpL in cells and in ligand blotsimplicates the N-terminal domain of apoB. Previous studies showed thatHTG-VLDL, but not normal VLDL S_(f)>60, bind with high affinity tocells, cause lipid accumulation, and bind to MBP200 and MBP235. HTG-VLDLsubfractions from subjects with elevated plasma triglyceride (>150mg/dl) contain more apoB-48 than VLDL subfractions from subjects withnormal plasma triglycerides (<150 mg/dl) after purification bycumulative flotation because of delayed chylomicron remnant clearance(Bradley et al., 1984). Taken together, these results suggest thatapoB-48 may be a preferred ligand, or at least contain a preferredconformational domain of apoB that enhances binding to this receptor.Thus, chylomicron subfractions isolated 4 h after a standardized fatload was studied (Weintraub et al., 1987). Chylomicrons, that is theTGRLP of S_(f)>400, were purified further by cumulative flotation intomore homogeneous subfractions of S_(f)>3200 (CM I), S_(f)1100-3200 (CMII), and S_(f)400-1100 (CM III) (Lingren et al., 1972). The largest twochylomicron fractions (CM I and II) contained apoB-48 as the onlydetectable apoB species (FIG. 15, lanes 1-4) whereas the smallestfraction (CM III) (lanes 5, 6) contained both apoB-48 and apoB-100.Immunochemical blotting (FIG. 15), allowed estimation that <0.1%, or <1in 1,000 particles, contain apoB-100 in the S_(f)>1100 subfractions.Lane 7 contains a typical fasting HTG-VLDL S_(f)100-400, with apoB-48 aswell as apoB-100 and apoE. All chylomicron subfractions containedimmunochemically detectable apoE (FIG. 15) and apoCs.

[0167] The three chylomicron subfractions were then tested for bindingto MBP200 and MBP235 and to the partially purified bovine LDL receptorby ligand blotting analysis; a representative experiment is shown inFIG. 16. All of the chylomicron subfractions, added at equivalentconcentrations, bound with high affinity to MBP200 and MBP235 (lanes 1,3, 5) as well as to the LDL receptor (lanes 2, 4, 6). Since apoB-48 isthe only apoB species immunochemically detectable in the largest twochylomicron subfractions (CN I and CM II) (FIG. 15), this stronglyimplicates apoB-48, or an apoB-48 domain, as the primary apoproteinbinding determinant for the distinct human apoE- and lipoproteinlipase-independent monocyte-macrophage receptor for TGRLP and itscandidate receptor proteins, MBP200 and MBP235. Indeed, the binding ofCM II to MBP200 and MBP235 was ˜90% inhibited by anti-apoB IgGs, but notby nonimmune IgG (FIG. 17).

EXAMPLE 23

[0168] Cloning of the Full Length cDNA of ApoB48 Receptor

[0169] MBP200 and MBP235 appear to be receptors for apoB48. To clone thecDNA for the human apoB48 receptor, designated apoB48R, nesteddegenerate oligonucleotide primers {bp 2763-2745,CCNCCNGTNACCATNARNC(SEQ ID No. 12) with 2048-fold degeneracy; and bp2754-2738, ACCATNARNCCYTCNGC (SEQ ID No. 13) with 256-fold degeneracybased on the unambiguous 10-residue sequence {AEGLMVTGGR (SEQ ID No.11)} previously used to produce receptor-specific antibodies (19), andthe λgt10 forward primer (AGCAAGTTCAGCCTGGTTAAG (SEQ ID No. 14) wereused to prime nested polymerase chain reactions (PCR) with a human THP-1monocyte cDNA library (Clontech) as a template. Based on the sequence ofthe 137 bp product, new primers {bp 2763-2745, CCCCCCGICACCATGAG (SEQ IDNo. 15) and bp 2754-2738, ACCATGAGRCCCTCTGC (SEQ ID No. 16)} were usedin PCR with the λgt10 forward arm primer to produce a 631 bp product(pcr-631) which was subcloned and sequenced.

[0170] The GeneAmp kit with AmpliTaq polymerase (Perkin Elmer) or theAdvantage-GC kit with KlenTaq polymerase (Clontech) was used for PCRexperiments. Initial cDNA clones were sequenced using the Sequenase 2.0kit (US Biochemicals); most were sequenced using the Dye TerminationCycle Sequencing kit (ABI/Perkin Elmer) in the automated sequencingcores at the University of Alabama at Birmingham (UAB). The cDNA clonesidentified by phage hybridization screening were sequenced directly orwere first subcloned into EcoRI-digested pBluescript II sk (Stratagene).PCR-generated clones were ligated into the TA cloning vector (PCR 2.1,Invitrogen) for sequencing. Both strands of all clones were sequenced;some regions were verified with different sequencing primers,particularly in GC-rich regions.

[0171] To screen the human THP-1 λgt10 cDNA library, the plasmidcontaining pcr-631 was digested with EcoRI and the agarose gel-purifiedpcr-631 (1 μg) was labeled by random priming using the Geniusdigoxigenin (DIG) labeling system (Boehringer Mannheim). The librarylysate was plated according to Clontech's protocol and plaques werelifted onto nylon filters. Filters were processed with the DIG-labeledpcr-631 according to the manufacturer's protocol and positive plaqueswere detected by chemiluminescence using the Lumiphos 530 reagent andexposed to Fuji NR film.

[0172] Antisense primers were based on the 5′-end sequence of theTHP-1×λ73 clone {bp 2203-2187, CCAGCCTCTTCTGGATG (SEQ ID No. 17) and2139-2123, TCTCCCAGTGACGGGCA (SEQ ID No. 18)}. Nested PCR using theseand the λgt10 forward arm primer and a human placenta λgt10 cDNA library(Clontech) as template produced a 1465 bp product which was cloned andsequenced (clone pcr-4-3).

[0173] Antisense primers based on the 5′ end sequence of clone pcr-4-3{722-699, CCTCAGTTTCCCCTGCATCTGCCT (SEQ ID No. 19) and PCR with gt10forward primer produced a 722 bp product (pcr-722) which was cloned andsequenced. Primers based on the sequence were used in PCR to amplify 5′end cDNA clones from the THP-1 cDNA library and THP-1 genomic DNA. Bothclones pcr-4-3 and pcr-722 were used as probes for phage hybridizationscreening of the human placenta λgt10 cDNA library. Six positive cloneswere sequenced and used to confirm the sequence of the PCR clones.

EXAMPLE 24

[0174] The apoB48R cDNA

[0175] The full length THP-1 monocyte cDNA (SEQ ID No. 1) (GenBankaccession #AF141332) is 3,744-basepairs (bp) with a Kozak start site (bp5-14), a 3264 bp ORF beginning at bp 11, and a stop codon (TGA) at bp3275 (FIG. 21). The 3′ untranslated region contains an Alu-likesequence, two polyadenylation signals (AATAAA) and a poly A tail (FIG.21). Sequencing multiple clones from both placenta and THP-1 monocytesspanning the full-length sequence indicated the human THP-1 monocyte andplacenta (GenBank accession #AF141334) sequences differ at 6 bases(mutations or gene polymorphisms); 3 of these predict identical aminoacids (b 520, A to G; b 574, A to G; b 1084, A to T), one is in the 3′untranslated region (b 3446, T to G), one causes a conservative aminoacid (aa) change (b 1434, G to A; R to K, aa residue 475), and oneintroduces a premature stop codon at aa 1077 (b 3239, G to T) that wouldcause a 12 residue truncation at the C-terminus in the placentalreceptor.

EXAMPLE 25

[0176] FISH Analysis of the apoB48R Gene

[0177] Normal human metaphase and interphase nuclei were prepared fromphytohaemagglutinin (PHA)-stimulated cultured blood lymphocytes usingstandard procedures. A cDNA insert was cut from a full length apoB48RcDNA which corresponded to base pairs bp 1 to 2614 of the cDNA clonelacked the 3′ untranslated region to eliminate the Alu-like sequence.The probe was labeled with Cy3-dUTP (Amersham) by nick translation, andthe chromosomal location was determined by FISH, as described previouslywith slight modification {T. Stokke et al., Genomics 26, 134 (1995)}.Briefly, 200 ng of Cy3-labeled cDNA was co-precipitated in the presenceof 1 μg of human Cot 1 DNA (Life Technologies) and hybridized to normalmetaphase and interphase nuclei. The cells were then counterstained withDAPI for chromosome identification. Fluorescent images were capturedwith digitized image microscopy as described [M. Sakamoto et al.,Cytometry 19, 60 (1995)]. The apoB48R gene (GenBank accession #AF141333)is located on chromosome 16p.1 as shown in FIG. 22.

EXAMPLE 26

[0178] apoB48R Genomic DNA

[0179] As only one chromosomal site was identified, there appear to beno closely related or duplicated apoB48R genes elsewhere in the humangenome. PCR cloning and sequencing of human THP-1 genomic DNA identifiedthree small introns within the coding sequence (Table 2). TABLE 2ApoB48R intronic sequences and location identified by PCR cloning usingTHP-1 monocyte- macrophage genomic DNA as template. Double underliningindicates intron-exon boundary motif GT . . . AG. Intron 1 (360 bp)between bp 67 and 68 of the cDNA, FIG. 21GTGAGAAGGGCAGACAGCTGCCAGATACTTGCACCCCATTCCCTGGGGCCTCACTTCCGGGCACCTCCCCTGGGGCCTCACCTTTCCCCTCCTCCTTCTGATCTCCTCTAACTGGAGATTGCTTTCTCAGGTTCAGGCAGACTCCTGGCCTAATATTTTCTGAATTTCAGTCCCCACCTCCAACCATGCGTCCTCGTACCCCTAATCGATGCCCCTTCTGGCTCCTTCTGCAAATCCTCTTCTTCTCCTTTCAGATCCCAGTACCCTCTTCCTTAACCTGGGCTCCTCCAGCCAGGGCCCCCAGGGAAAGGGCTGGGACTCTCCTCAATGACTCTCCCCTCTCTCTCTCTTTTTTCCTAG  (SEQ ID No. 20) Intron 2(84) between bp 3228 and 3229 of the cDNA, FIG. 21GTGAGGGCTCTTGGTGGGGTCTCGGGGGGAACGAGTGGAATCCCGAAGCCGGCCCCATGGTCCTCTGTGCCCCCTTTCCTGCAG  (SEQ ID No.21) Intron 3 (86) between bp 3207 and3208 of the cDNA, FIG. 21GTGAGGGCTCTTGGTGGGGTCTCGGGGGGAACGAGTGGAATCCCGAAGCCGGCCCCATGGTCCTCTGTGCCCCCTTTCCTGCAG  (SEQ ID No.22)

EXAMPLE 27

[0180] apoB48R Genomic DNA

[0181] The 1088 amino acid deduced sequence (SEQ ID No. 2) of the THP-1monocyte apoprotein contains three internal sequences previouslyobtained by microsequence analysis of tryptic digests of the purifiedreceptor (FIG. 21). Immunoblotting data and transfection studiespresented below indicate this cDNA is sufficient to encode a functionalapoB48R. The sequence is unique; moreover, in that it is not closelyrelated to any known protein. Unlike other lipoprotein receptors, whichhave functionally important cysteine-rich domains, there are only eightcysteines distributed throughout the apoB48R sequence. These may beinvolved in internal disulfides, giving rise to the observedmicroheterogeneity of unreduced receptor extracts observed on SDS-PAGEthat disappears upon reduction (17, 21). The reduced and nonreducedforms of the receptor have full ligand binding activity, indicating thatthe cysteines are not directly involved in ligand binding.

[0182] The apoB48R protein is highly polar, with 242 negative and 122positive amino acids, and contains only two hydrophobic regions that arepotential membrane spanning or lipid-interacting domains. The firsthydrophobic region, amino acids 1 to 30, is predicted to be helical bysome but not all analyses, contains a putative leader sequence, aleucine zipper motif (amino acids 8 to 29), and a hydrophobic domainencompassing three-fourths of the helical face. The other hydrophobicdomain (amino acids 751 to 773) is 23-residues long and has a highhelical potential, consistent with known transmembrane domains.

[0183] Posttranslational glycosylation or other modification mightaccount for the disparity between the deduced (˜114.8 kD) and theapparent M_(r) determined by SDS-PAGE (˜200 kD). A potentialglycosaminoglycan attachment site is at amino acid 118, a potentialN-glycosylation site is at amino acid 617, and 4 possibleO-glycosylation sites are at amino acids 265, 514, 565 and 942. However,treatment of partially purified receptor preparations with variousglycosidases failed to affect significantly the mobility of the apoB48Ron SDS-PAGE or its ligand binding properties (data not shown),suggesting that it contains little, if any, functionally significantcarbohydrate.

[0184] ApoB48R does not contain a tyrosine-based internalization signallike that in the LDLR family. Rather, it contains tyrosine-independentdi-leucine motifs (ExxxLL) (FIG. 21), which signal coated pitlocalization and internalization in immune cells (Mellman, R., 1996). Asthe apoB48R functions as a receptor for uptake of TGRLP when transfectedinto CHOs as described below, one of these di-leucine motifs may performthis function. The significance of the internal, unique repeatingelement (3 repeats of the 9 residue sequence GGEEAETAS and 8 nonexactrepeats) is not known.

[0185] Repeated attempts to dissociate polymeric species failed to shiftthe mobility of the apoB48R from ˜200 kD Mr, including boiling andreducing (Ramprasad, M. P., et al., 1995). There are, however, threepotential coiled-coil domains in the predicted protein sequence thatcould promote homodimerization of the apoB48R and account for the sizediscrepancy (FIG. 21). Other than the motifs noted above, the apoB48Rprotein has no other common homologies to known proteins represented inGenBank.

EXAMPLE 28

[0186] Evidence for Homodimerization of apoB48R

[0187] Homodimerization is supported by results withglutathione-S-transferase (GST)-fusion proteins. GST fusion proteins ofthree domains encoded by clone pcr-4-3, 73-3, and pcr-631 wereconstructed in the pGEX-5X-3 vector (Pharmacia) according to themanufacturer's instruction. cDNA was digested from the vectors withEcoRI and ligated into the EcoRI-digested pGEX-5X-3. Transformants werescreened for production of the appropriate fusion product and theplasmid DNA was sequenced to verify that the clones were in the correctorientation and reading frame for expression. Fusion proteins wereproduced by batch culture (24 L) of selected transformants and purifiedaccording to the manufacturer's protocol on glutathione-Sepharose.

[0188] The fusion proteins were used as immunogens to generatepolyclonal antibodies in rabbits by standard techniques. IgG wereprecipitated from antisera with (NH₄)₂ SO₄, reconstituted in PBS, storedat 4%C (short term), and demonstrated specificity for all forms of theapoB48R (MBP200, MBP235, and MBP200R) by immunoblotting.

[0189] Homodimerization is supported by the results with theglutathione-S-transferase (GST)-fusion protein containing amino acid 223to 710. This fusion protein migrates as a dimer when analyzed onSDS-PAGE, suggesting spontaneous dimerization of this domain (27). Incontrast, GST fusion proteins from more C-terminal domains of theapoB48R that lack potential coiled-coil domains (amino acid 629-1088 andamino acid 714-917) do not dimerize but instead migrate as expected bytheir predicted Mrs.

EXAMPLE 29

[0190] Analysis of apoB48 Expression.

[0191] Expression of apoB48 was analyzed in multiple tissues using areverse transcriptase PCR technique. Total RNA was isolated from cellsusing the single-step protocol (Ausubel et al., 1995). Oligonucleotideprimers were synthesized corresponding to bp 2756-2736 (antisense) andbp 2185-2204 (sense). First strand cDNA synthesis was performed on 5 μgof total RNA from each cell type primed with the antisense primer andSuperscript II reverse transcriptase (Gibco/BRL). The subsequent PCRamplification used 10 pmol of the antisense primer, 10 pmol of the senseprimer, and 20 μL of the first strand cDNA. The PCR products wereanalyzed b y electrophoresis on a 1% agarose.

[0192] PCR screening of a human tissue cDNA panel with gene-specificprimers identified the receptor cDNA in the brain, heart, kidney, liver,lung, pancreas, and placenta but not in skeletal muscle (FIG. 23). Thelevel of receptor cDNA appeared to be greatest in lung and placenta asthese were the only positive tissues after 25 PCR cycles. Controlreactions with GPDH probes or primers verified approximately equalrepresentation of transcripts in the mRNA and cDNA panels. Not shown areRT-PCR data indicating the expression of the apoB48R mRNA in humanblood-borne, THP-1, and U-937 monocyte-macrophages; in murine P388D₁macrophages and NIH-3T3 cells (thought to be of EC origin); and in humanumbilical vein ECs. CHOs, human fibroblasts, and HepG2s were negative,consistent with previous studies (Gianturco, S.H., et al., 1988 and1994c that indicate that apoB48R is expressed primarily by cells ofreticuloendothelial origin.

EXAMPLE 30

[0193] Transfection Studies

[0194] Transfection studies used THP-1 monocyte apoB48R cDNA includingthe first intron. The minigene was constructed by ligation of a 2454 bpPCR-generated genomic clone (bp 2-2095 of cDNA) that includes the firstintron (Table 2) with the λ73 clone after digestion with EagI. Theminigene was removed from its pBluescriptII KS (−) vector by EcoRI/NotIdigestion and ligated into pcDNA 3.1 (−) with Ready-to-go T4 ligase(Pharmacia) according to the manufacturer's procedure. Clones wereisolated by standard procedures and verified by partial DNA sequencingas described.

[0195] The minigene was transfected into CHO-KI cells (ATCC) usinglipofectamine (Gibco) according to the manufacturer's recommendationsfor adherent cells, and cells were selected in medium containing G418(0.5 mg/ml; Agribio). After two-four weeks periods in selection medium,cells were harvested by trypsin treatment and subcultured ontocoverslips (12 mm) for visualization experiments (Oil red O staining andDiI) or into 35 mm dishes for measurement of lipid accumulation afterincubation with lipoproteins at the levels and times given for eachexperiment.

[0196] Transfection of full length apoB48R cDNA constructs (with andwithout the first intron) into CHO-K1 cells conferred apoB48R expressionand activity, whereas transfection with vector alone or inverted cDNAinsert did not. Ligand and western blotting experiments performed aspreviously described (Gianturco, S. H., et al., 1994c and 1998) documentexpression of a single apoB48R species in the R-transfected cells, butnot in vector-transfected cells, that exhibits coincident ligand bindingactivity and apoB48R immunoreactivity (FIG. 24).

[0197] The apparent Mr of 190 kD in the R-transfected CHOs is slightlylower than the active forms in THP-l's (200 and 235 kD). The Mrdifferences and the presence of a single rather than two ligand bindingspecies are likely due to differences in processing in CHO cells versusmacrophages. Of note, however, the apparent M_(r) of the receptor in theapoB48R-transfected CHOs approaches twice that predicted by the cDNA,consistent with homodimerization of the receptor via protein interactingmotifs previously noted.

EXAMPLE 31

[0198] Analysis of TGRLP Uptake

[0199] ApoB48R-mediated TGRLP uptake by transfected CHOs was determinedby three alternative methods: first, by TGRLP-induced increases incellular TG mass (FIG. 25), a sensitive, quantitative endpoint since TGis the predominant lipid in the lipoprotein and thus the predominantlipid accumulated by cells. The second method is by the rapid andmassive TGRLP-induced accumulation of large Oil Red O-positivecytoplasmic lipid droplets that form after lysosomal hydrolysis andreesterification (Gianturco, S. H., et al., 1982 and 1988) (FIG. 26).Finally, the third method is b y uptake of TGRLP labeled with thelipid-soluble fluorescent dye1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchloratevisualized by fluorescence microscopy (data not shown).

[0200] When transfected cells are incubated with increasingconcentrations of tryp-VLDL (Gianturco, S. H. et al., 1986) for 4 hoursat 37° C. (FIG. 25A), apoB48R-transfected cells show a rapid andcurvilinear accumulation of TG, indicating saturable R-mediated uptake.In contrast, vector-transfected cells show only linear, nonspecificuptake. Similar results were obtained in multiple experiments withtryp-VLDL devoid of apoE, chylomicrons containing apoB48 as the onlyapoB species, and HTG-VLDL containing apoB48 and apoB100, but no uptakewas with normal VLDL containing apoB100 as the only detectable apoBspecies (FIG. 25B). G418-selected CHOs transfected with an apoB48Rminigene containing the first intron show massive, rapid accumulation oflarge Oil Red O-positive lipid droplets when incubated for about 3 hourswith tryp-VLDL devoid of apoE or with chylomicrons that contain apoB48but not apoB100 (FIG. 26). This is not seen with CHOs containing vectoralone.

[0201] These experiments (each replicated 3 to 10 times) indicate bythree alternative endpoints that the protein encoded b y this cDNA issufficient to confer apoB48R activity on the transfected cells similarthat observed in human monocyte-macrophages in ligand specificity andkinetics of uptake (Gianturco, S. H., et al., 1994c and 1998). Thesetransfection studies demonstrate unequivocally that the apoB48R bindsand internalizes TGRLP when out of the context of the macrophage, i.e.,in the absence of other macrophage-specific proteins such as apoE orlipoprotein lipase that can enhance macrophage uptake of lipoproteins.Thus the apoB48-specific receptor activity, previously documented byboth cell and ligand blotting studies in human blood-borne and THP-1monocytes and macrophages, can indeed be attributed to this new andunique receptor protein encoded by this cDNA and not to a combination ofother macrophage processes.

EXAMPLE 32

[0202] ApoB48R and Atherosclerotic Plaques

[0203] Excessive uptake of TGRLP by the apoB48R may promoteatherosclerosis in vivo. To test this hypothesis, immunohistochemicalstudies were undertaken to determine if apoB48R is expressed inatherosclerotic plaques. Immunohistochemical staining used the samepolyclonal antibodies against a domain of the apoB48R (amino acid 223 to710) (27) which were used for immunoblotting (FIG. 24) and a labeledstreptavidin-biotin kit (Nichirei Co., Tokyo) according tomanufacturer's instruction. Paraffin sections were treated with xylene,then ethanol, and finally immersed in 0.3% H₂O₂ in methanol for 45 minto block endogenous peroxidase activity. All specimens were rinsed withphosphate buffered saline (PBS, pH 7.4) and incubated with 10% normalgoat serum for 12 h at 4° C. The antibody used for staining wasanti-apoB48R polyclonal rabbit IgG (1:1000) directed against the GSTfusion protein linked to aa 223 to 710 of the apoB48R. Preimmune rabbitIgG was used as a negative control. To detect macrophages inatherosclerotic lesions, a monoclonal antibody against human macrophageswas used (HAM56; Dako, Glostrup, Denmark). The specimens were incubatedovernight with each primary antibody, rinsed 3× with PBS, and incubatedwith a biotinylated horse anti-mouse or anti-rabbit IgG for 30 min atroom temperature. The specimens were rinsed 3 times with PBS, incubatedwith peroxidase-conjugated streptavidin for 30 min and visualized usinga 3-amino-9-ethylcarbazole (AEC) substrate (Nichirei, Tokyo), andstained with hematoxylin.

[0204]FIG. 27 shows representative serial sections of a carotid arteryatheroma. The macrophage-specific monoclonal antibody HAM56 identifiesmacrophages and macrophage foam cells around the lipid core.Anti-apoB48R IgGs bind to macrophages and foam cells on the shoulder ofthe plaque. Preimmune IgGs do not bind to any cells in the lesion. Foamcells in early, aortic fatty streak lesions as well as in more advancedcoronary lesions were also apoB48R-positive (35), supporting thepossibility that the apoB48R may contribute to foam cell formation andatherogenesis in vivo.

SUMMARY

[0205] The present invention discloses the ligand for a novel receptorand identification of the minimal requirements for receptor binding.Therefore, this invention describes means by which agents can bedelivered to specific cells expressing the receptor or means b y whichthe entry of ligands may be regulated for therapeutic use.Triglyceride-rich lipoprotein ligands of this receptor, in addition tocausing lipid accumulation in monocytes and macrophages, cause increasedmonocyte adhesion to endothelial cells in vitro—a potentiallyatherogenic effect—by inducing the expression of adhesion molecules onboth endothelial cells and on monocytes; cause phenotypic conversion ofendothelial cells into multinucleated giant cells, like those found overhuman coronary atherosclerotic lesions; impair endothelial cell-mediatedfibrinolysis; and induce enhanced expression of tissue factor mRNA andantigen in endothelial cells and monocytes, a prothrombotic effect.

[0206] Lipoproteins bind to this receptor via an apoB domain within theamino-terminal region. Competitive cell and ligand blotting studiesdemonstrate that anti-apoB, but not anti-apoE, anti-CII or nonimmuneIgG, inhibit binding of triglyceride-rich lipoproteins both to THP-1sand to the receptor proteins. Lactoferrin (100 μg/ml), heparin (10mg/ml), and the human receptor associated protein do not inhibittriglyceride-rich lipoprotein binding. In contrast, lipoprotein lipase,which is known to bind to an N-terminal domain of apoB (Choi et al.,1995), partially inhibits binding of triglyceride-rich lipoproteins inboth cell and ligand blotting studies via its interaction with apoB.Plasma chylomicrons S_(f)1100-3200 (4 h postprandial) that containapoB-48 as the only apoB species bind specifically to the receptorproteins, and this can be inhibited b y anti-apoB antibodies. Lipolysisof these S_(f)>1,100 apoB-48 particles diminishes binding to thereceptor particles proteins (MBP200 and MBP235), while enhancing bindingto the low density lipoprotein receptor. Smaller apoB-100 containinglipoprotein (intermediate density lipoprotein and low densitylipoprotein) and normal very low density lipoprotein binding to thereceptor on ligand blots can b e detected. Competitive binding studiesin monocyte-macrophages and in ligand blots, however, indicate thebinding is of lower affinity than that of hypertriglyceridemictriglyceride-rich lipoproteins of S_(f)>60 and chylomicrons and theirremnants, since twenty- to forty-fold excess of these lipoproteins donot inhibit binding of the primary lipoprotein ligands (Gianturco,1994). However, binding to MBP200 and MBP235 of low density lipoproteinthat contains apoB-100 as the only detectable apo protein indicates thatapoB is sufficient to mediate binding. These data strongly support thatthe monocyte-macrophage triglyceride-rich lipoprotein receptor-bindingdomain is within the N-terminal half of apoB corresponding to apoB-48 ator near the lipoprotein lipase binding domain and not in a heparinbinding domain. These data also suggest that these receptors couldaccount for the observed rapid peripheral macrophage uptake of plasmachylomicrons and represent a new apoB and monocyte-macrophage (andrelated cell type)-specific apoB receptor that is important in thenutrition of receptor-expressing cells, including circulating monocytes,accessible macrophages and placenta, especially in the postprandialstate.

[0207] Knowledge of the receptor-binding determinants in the naturalligands (and knowledge of the domain(s) in the receptor that bind theligands, i.e., the ligand-binding domains) provides an individual havingordinary skill in this art with the knowledge to design agents for thecell-specific delivery of drugs, vitamins, genes, enzymes, hormones,cytokines, growth factors, or inhibitors to cells expressing thereceptor, including monocytes, macrophages, endothelial cells, placentalcells, astrocytes, and the tissues which contain these cells, includingbone marrow, blood vessels, spleen, lymph nodes, tonsils, appendix,thymus, liver, placenta, brain. For example, determination of theminimal peptide sequence sufficient to bind to the receptor anddetermination of the domain in the receptor which binds the ligand canbe used to design synthetic peptides for incorporation in liposomescontaining the desired chemical, enzyme, or gene. Alternatively, theagent could be directly linked to the peptide or to antibodies againstthe receptor. Such strategies could be useful for: (1) the treatment ofmonocytic leukemia; (2) the treatment of tuberculosis and otherdiseases, such as leprosy, caused by pathogenic mycobacteria, whichrequire uptake by macrophages for pathogenesis (Schorey et al., 1997);(3) the treatment of AIDS, since HIV is harbored in macrophages aftereradication from lymphocytes; (4) the inhibition or enhancement ofangiogenesis (to inhibit angiogenesis in tumors or to promoteangiogenesis in wound healing or after infarcts); (5) the inhibition orenhancement of fibrinolysis to promote clot formation or dissolution;(6) enhancement or inhibition of tissue factor production; and (7) thedelivery of agents to the developing embryo. Conversely, syntheticagents which bind to the receptor and block or inhibit lipoprotein orother ligand binding to the receptor may also be useful in preventingexcess uptake of lipoproteins/ligands via this route, thereby inhibitingfoam cell formation and increased monocyte adhesion to endothelialcells, and decreasing inhibition of fibrinolysis by VLDL and inhibitionof uptake of lipoprotein-soluble agents that are pathogenic for aspecific cell or tissue, etc. Receptor mutations may be associated withspecific dyslipidemias or abnormal postprandial triglyceride metabolism,which in turn are linked to increased risk of cardiovascular disease.Tests for receptor structure/function could be used to identify subjectsat increased risk due to receptor defects. Abnormalities in the ligandsof the receptor could also lead to defective or enhanced binding anduptake via this receptor and may also be predictive of increased diseaserisk. Receptor abnormalities may be involved in placental pathologies.

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[0370] Any patents or publications mentioned in this specification areindicative of the levels of those skilled in the art to which theinvention pertains. These patents and publications are hereinincorporated by reference to the same extent as if each individualpublication was specifically and individually indicated to beincorporated by reference.

[0371] One skilled in the art will readily appreciate that the presentinvention is well adapted to carry out the objects and obtain the endsand advantages mentioned, as well as those inherent therein. The presentexamples along with the methods, procedures, treatments, molecules, andspecific compounds described herein are presently representative ofpreferred embodiments, are exemplary, and are not intended aslimitations on the scope of the invention. Changes therein and otheruses will occur to those skilled in the art which are encompassed withinthe spirit of the invention as defined by the scope of the claims.

[0372]

1 22 1 3773 DNA Homo sapien ApoB48R cDNA sequence 1 gcggccgcgtctaccgcggc cgcgtctacg acagacagga tggacttcct ccggctatac 60 ctccctgggctgcaccaggc cttgaggggg gcactggatt ccctcggcac ctttgtctcc 120 tacctcctgggagatgcagt ccccactgta gagcgggagg cgcaggcggc tgaggaactg 180 ggggtggtggcggtgggaaa gacagggaag attgtagagg aggaagccca ggaggacctg 240 gagggccttagaggcagcca aaacgagggg gctggaaggc tgagagggcc tggagatgac 300 agaagacatgaagtggggag ctcagctgta gaacagacct ggggctgggg agatggcagc 360 tcccatgggtcccaagcaga gaggcaggac agtggggctg gggagacagc caaggctgcc 420 aggtgccaggagccaagcgc ccacttggag gccagaaaga aatccaaggc agggtctggg 480 gcttgccaagacaggagcgg ccaagcccag gagaggcagg agtcccatga gcaggaagtg 540 aacagagaggagaggctgag aagctgggaa caggaggagg aggaggaaga ggtcagggca 600 agggagccagggatggccag aggggcggag tcagagtgga cctggcatgg ggagacggag 660 gggaaggctggtgctgttgg gccaaaggcg gcaggggaca accgggagat ggagcagggg 720 gtcagggaggcagatgcagg ggaaactgag gagcctgggg ccgaaggggc tgggaaagga 780 gaagaggtggtagtggtgga gaaggcctgt gaaagcacta gggcatgggg gacgtggggc 840 ccaggggcagagcctgagga ctggggaatc ttaggcagag aggaggccag gacaacccca 900 ggtagggaagaggccagggc aattttagat ggggaggaag ccaggacaat ctcaggcggg 960 gaggaggctgagacagcctc aggcggggag gaggctgaaa cagcctcagg cggggaggag 1020 gccgggacagcctcgggagg ggaggaggcc gggatatcct caggcgggga ggctgggaca 1080 gcctcaggaggggaggaggc cgggacagcc tctggagggg acgaggcctg gacaacctca 1140 ggcaaagaggaggctgacct gctgggagtc agacagactc aatatggagc agttccagga 1200 gaaaggctcctagaggctac tggaaaagtc tgggtcctag aggaggaggg ggatgaggag 1260 agagaggctgaggtgagccc tttccccaaa caggcccagg tcctgggcac tgaaagaaca 1320 gaagaggctgctgagagcca gaccgcaggg agggaagctg tgggaggcca ggaggcaggg 1380 gagagctttgagggccaggt agacctgcgt ggtaaggagg ctgagatgag gcaggacttg 1440 gggatcagggccgaccgggc caagatggaa gagctggtac aggcagagga ggcccaggag 1500 gagagagggagcagcaggga tccagtggct gagctgccct cagatggaga ggctgaaggc 1560 actgccgacttggaggcaac tccagaggcc aggcctgagg aggagctcac aggggaggag 1620 agtgaggcggcccagactag ctgtggccta ctgggcgtgg aatggggtgg cctcacacac 1680 agcgtcaccaaaggccaggg acctgagctg atggggggtg cccagacccc aactaagcaa 1740 cccgaggaaagggaggcagg ggaggtggag ctcatgggag ttctggccct gagcaaagag 1800 gagcaggagaggagcctgga ggcaggtccc aggcacgcgg ggtctgtaaa gcctgaggcc 1860 tccgaggccttcccaggagc ctgggaaaac cgcacgagaa aggacatgga gagaggaaat 1920 actcaggaggatgcggccga tggcgagcag cgggaggagg aggagactgc gggaggccag 1980 accctggcggctgaggctga aggagaccga gagtctgaac tatcagaagt cccagaggca 2040 ggcggggaggggctgacaac ccaggacgcg ggatgtggaa ctgaggaggg agaggcatct 2100 gtctcagagaaccaggagct ggacggaagc acaggggcag acgcagggcc ttgcccgtca 2160 ctgggagaggcctatgccag agaaactgag gatgaggagg cggaggctga cagaacatcc 2220 agaagaggctggaggctgca agcggtggct gtgggcctcc cggaccgtga ggatgcacag 2280 actggctctgtggctgctgg gattatgggg ggtgatgtgg tcccacacat cagcgctgct 2340 ggcgctggtgaagctttgga aggggcgctt gggcaaggct gggactcgaa agaaaaggaa 2400 gaggcagcagcaggagagca tgcaggtggg caagaatttg gtctggaggg ctcagcagag 2460 gaagaggtgactggcagagg cagccaagta gaggcttttg agtccaggga gggaggacct 2520 tggggagggcgggtagaggc cgaggaatct gcaggcgcag aggacagctg tgggctggat 2580 cccgcgggctcccagacagc gagggcagag gggatgggag ccatggtgga ggctgggggg 2640 cttctagaaaagtggacgct gttggaagaa gaggctgttg gatggcagga gagagaacag 2700 agggaagacagtgaggggcg gtgtggggac taccaccctg agggagaggc accaaggctc 2760 cttgatgcagagggtctcat ggtgaccggg ggccggaggg cagaggccaa ggagactgag 2820 ccagaaagcctggaacatgt caggggccag gaggagcagc caacacacca ggcccctgca 2880 gaagctgcgccggagtcagt cggggaagcc gagacggctg aggccatggg cagtgccaga 2940 ggaggtgctgccaacagctg gagcgaggcc ccgctccccg ggtccctcct agacgtctct 3000 gtcccaaggagtcgcgtgca cctctcgaga agctcctcac agcgtcgctc ccggccctct 3060 tttcgtcggactccggcctg ggagcagcag gaggagcccc cagcccccaa ccctcctgag 3120 gaggagctgtcagctcctga gcagagaccc ctccagctgg aggaacccct ggagccaagc 3180 cctctgaggcatgatgggac cccggtgcca gccaggagaa ggcccctggg acacgggttt 3240 ggcctcgcgcaccctggcat gatgcaggag ctgcaagccc gtctgggccg gcctaagccc 3300 cagtgactgagacccggtgc tctgggagcc aggccctgag tgggtgccag aaggcttgct 3360 ccaatgccactgagccctgc tccctctgcc actgtggaca catcctctcc accctctggg 3420 cctcagtgtcttgatgtatc attcatggag caggcaaaac cagacgtctg ggaataccgt 3480 gaacttaaggagtctgattc tccgacacag gctggtggac cacctacccc actgagacca 3540 cctctcagggtgcctgccct ggttcctccc cagcctgagt cagctgtctg gactgcaagg 3600 aggctgggcacgggggctca cgcctgtcac cccagagctt tgggaggcca aggtgggagg 3660 atcgcttgagaccaggagtt cgagaccagc ctgggcagca tagcaagatc cccatctttt 3720 aaaaacaaaataaaacaata aagactgcaa ggaaaaaaaa aaaaaaaaaa aaa 3773 2 1088 PRT Homosapien 2 Met Asp Phe Leu Arg Leu Tyr Leu Pro Gly Leu His Gln Ala Leu 510 15 Arg Gly Ala Leu Asp Ser Leu Gly Thr Phe Val Ser Tyr Leu Leu 20 2530 Gly Asp Ala Val Pro Thr Val Glu Arg Glu Ala Gln Ala Ala Glu 35 40 45Glu Leu Gly Val Val Ala Val Gly Lys Thr Gly Lys Ile Val Glu 50 55 60 GluGlu Ala Gln Glu Asp Leu Glu Gly Leu Arg Gly Ser Gln Asn 65 70 75 Glu GlyAla Gly Arg Leu Arg Gly Pro Gly Asp Asp Arg Arg His 80 85 90 Glu Val GlySer Ser Ala Val Glu Gln Thr Trp Gly Trp Gly Asp 95 100 105 Gly Ser SerHis Gly Ser Gln Ala Glu Arg Gln Asp Ser Gly Ala 110 115 120 Gly Glu ThrAla Lys Ala Ala Arg Cys Gln Glu Pro Ser Ala His 125 130 135 Leu Glu AlaArg Lys Lys Ser Lys Ala Gly Ser Gly Ala Cys Gln 140 145 150 Asp Arg SerGly Gln Ala Gln Glu Arg Gln Glu Ser His Glu Gln 155 160 165 Glu Val AsnArg Glu Glu Arg Leu Arg Ser Trp Glu Gln Glu Glu 170 175 180 Glu Glu GluGlu Val Arg Ala Arg Glu Pro Gly Met Ala Arg Gly 185 190 195 Ala Glu SerGlu Trp Thr Trp His Gly Glu Thr Glu Gly Lys Ala 200 205 210 Gly Ala ValGly Pro Lys Ala Ala Gly Asp Asn Arg Glu Met Glu 215 220 225 Gln Gly ValArg Glu Ala Asp Ala Gly Glu Thr Glu Glu Pro Gly 230 235 240 Ala Glu GlyAla Gly Lys Gly Glu Glu Val Val Val Val Glu Lys 245 250 255 Ala Cys GluSer Thr Arg Ala Trp Gly Thr Trp Gly Pro Gly Ala 260 265 270 Glu Pro GluAsp Trp Gly Ile Leu Gly Arg Glu Glu Ala Arg Thr 275 280 285 Thr Pro GlyArg Glu Glu Ala Arg Ala Ile Leu Asp Gly Glu Glu 290 295 300 Ala Arg ThrIle Ser Gly Gly Glu Glu Ala Glu Thr Ala Ser Gly 305 310 315 Gly Glu GluAla Glu Thr Ala Ser Gly Gly Glu Glu Ala Gly Thr 320 325 330 Ala Ser GlyGly Glu Glu Ala Gly Ile Ser Ser Gly Gly Glu Ala 335 340 345 Gly Thr AlaSer Gly Gly Glu Glu Ala Gly Thr Ala Ser Gly Gly 350 355 360 Asp Glu AlaTrp Thr Thr Ser Gly Lys Glu Glu Ala Asp Leu Leu 365 370 375 Gly Val ArgGln Thr Gln Tyr Gly Ala Val Pro Gly Glu Arg Leu 380 385 390 Leu Glu AlaThr Gly Lys Val Trp Val Leu Glu Glu Glu Gly Asp 395 400 405 Glu Glu ArgGlu Ala Glu Val Ser Pro Phe Pro Lys Gln Ala Gln 410 415 420 Val Leu GlyThr Glu Arg Thr Glu Glu Ala Ala Glu Ser Gln Thr 425 430 435 Ala Gly ArgGlu Ala Val Gly Gly Gln Glu Ala Gly Glu Ser Phe 440 445 450 Glu Gly GlnVal Asp Leu Arg Gly Lys Glu Ala Glu Met Arg Gln 455 460 465 Asp Leu GlyIle Arg Ala Asp Arg Ala Lys Met Glu Glu Leu Val 470 475 480 Gln Ala GluGlu Ala Gln Glu Glu Arg Gly Ser Ser Arg Asp Pro 485 490 495 Val Ala GluLeu Pro Ser Asp Gly Glu Ala Glu Gly Thr Ala Asp 500 505 510 Leu Glu AlaThr Pro Glu Ala Arg Pro Glu Glu Glu Leu Thr Gly 515 520 525 Glu Glu SerGlu Ala Ala Gln Thr Ser Cys Gly Leu Leu Gly Val 530 535 540 Glu Trp GlyGly Leu Thr His Ser Val Thr Lys Gly Gln Gly Pro 545 550 555 Glu Leu MetGly Gly Ala Gln Thr Pro Thr Lys Gln Pro Glu Glu 560 565 570 Arg Glu AlaGly Glu Val Glu Leu Met Gly Val Leu Ala Leu Ser 575 580 585 Lys Glu GluGln Glu Arg Ser Leu Glu Ala Gly Pro Arg His Ala 590 595 600 Gly Ser ValLys Pro Glu Ala Ser Glu Ala Phe Pro Gly Ala Trp 605 610 615 Glu Asn ArgThr Arg Lys Asp Met Glu Arg Gly Asn Thr Gln Glu 620 625 630 Asp Ala AlaAsp Gly Glu Gln Arg Glu Glu Glu Glu Thr Ala Gly 635 640 645 Gly Gln ThrLeu Ala Ala Glu Ala Glu Gly Asp Arg Glu Ser Glu 650 655 660 Leu Ser GluVal Pro Glu Ala Gly Gly Glu Gly Leu Thr Thr Gln 665 670 675 Asp Ala GlyCys Gly Thr Glu Glu Gly Glu Ala Ser Val Ser Glu 680 685 690 Asn Gln GluLeu Asp Gly Ser Thr Gly Ala Asp Ala Gly Pro Cys 695 700 705 Pro Ser LeuGly Glu Ala Tyr Ala Arg Glu Thr Glu Asp Glu Glu 710 715 720 Ala Glu AlaAsp Arg Thr Ser Arg Arg Gly Trp Arg Leu Gln Ala 725 730 735 Val Ala ValGly Leu Pro Asp Arg Glu Asp Ala Gln Thr Gly Ser 740 745 750 Val Ala AlaGly Ile Met Gly Gly Asp Val Val Pro His Ile Ser 755 760 765 Ala Ala GlyAla Gly Glu Ala Leu Glu Gly Ala Leu Gly Gln Gly 770 775 780 Trp Asp SerLys Glu Lys Glu Glu Ala Ala Ala Gly Glu His Ala 785 790 795 Gly Gly GlnGlu Phe Gly Leu Glu Gly Ser Ala Glu Glu Glu Val 800 805 810 Thr Gly ArgGly Ser Gln Val Glu Ala Phe Glu Ser Arg Glu Gly 815 820 825 Gly Pro TrpGly Gly Arg Val Glu Ala Glu Glu Ser Ala Gly Ala 830 835 840 Glu Asp SerCys Gly Leu Asp Pro Ala Gly Ser Gln Thr Ala Arg 845 850 855 Ala Glu GlyMet Gly Ala Met Val Glu Ala Gly Gly Leu Leu Glu 860 865 870 Lys Trp ThrLeu Leu Glu Glu Glu Ala Val Gly Trp Gln Glu Arg 875 880 885 Glu Gln ArgGlu Asp Ser Glu Gly Arg Cys Gly Asp Tyr His Pro 890 895 900 Glu Gly GluAla Pro Arg Leu Leu Asp Ala Glu Gly Leu Met Val 905 910 915 Thr Gly GlyArg Arg Ala Glu Ala Lys Glu Thr Glu Pro Glu Ser 920 925 930 Leu Glu HisVal Arg Gly Gln Glu Glu Gln Pro Thr His Gln Ala 935 940 945 Pro Ala GluAla Ala Pro Glu Ser Val Gly Glu Ala Glu Thr Ala 950 955 960 Glu Ala MetGly Ser Ala Arg Gly Gly Ala Ala Asn Ser Trp Ser 965 970 975 Glu Ala ProLeu Pro Gly Ser Leu Leu Asp Val Ser Val Pro Arg 980 985 990 Ser Arg ValHis Leu Ser Arg Ser Ser Ser Gln Arg Arg Ser Arg 995 1000 1005 Pro SerPhe Arg Arg Thr Pro Ala Trp Glu Gln Gln Glu Glu Pro 1010 1015 1020 ProAla Pro Asn Pro Pro Glu Glu Glu Leu Ser Ala Pro Glu Gln 1025 1030 1035Arg Pro Leu Gln Leu Glu Glu Pro Leu Glu Pro Ser Pro Leu Arg 1040 10451050 His Asp Gly Thr Pro Val Pro Ala Arg Arg Arg Pro Leu Gly His 10551060 1065 Gly Phe Gly Leu Ala His Pro Gly Met Met Gln Glu Leu Gln Ala1070 1075 1080 Arg Leu Gly Arg Pro Lys Pro Gln 1085 3 631 DNA Homosapien 3 aagctgttgt atgggtcaga gaaactgagg atgaggaggc ggaggctgacagaacatcca 60 gaagaggctg gaggctgcaa gcggtggctg tgggcctccc ggaccgtgaggatgcacaga 120 ctggctctgt ggctgctggg attatggggg gtgatgtggt cccacacatcagcgctgctg 180 gccgtggtga agctttggaa ggggcgcttg ggcaaggctg ggactcgaaagaaaaggaag 240 aggcagcagc aggagagcat gcaggtgggc aagaatttgg tctggagggctcagcagagg 300 aagaggtgac tggcagaggc agccaagtag aggcttttga gtccagggagggaggacctt 360 ggggagggcg ggtagaggcc gaggaatctg caggcgcaga ggacagctgtgggctggatc 420 ccgcgggctc ccagacagcg agggcagagg ggatgggagc catggtggaggctggggggc 480 ttctagaaaa gtggacgctg ttggaagaag aggctgttgg atggcaggagagagaacaga 540 gggaagacag tgaggggcgg tgtggggact accaccctga gggagaggcaccaaggctcc 600 ttgatgcaga gggactcatg gtgacggggg g 631 4 209 PRT Homosapien 4 Ala Val Val Trp Val Arg Glu Thr Glu Asp Glu Glu Ala Glu Ala 510 15 Asp Arg Thr Ser Arg Arg Gly Trp Arg Leu Gln Ala Val Ala Val 20 2530 Gly Leu Pro Asp Arg Glu Asp Ala Gln Thr Gly Ser Val Ala Ala 35 40 45Gly Ile Met Gly Gly Asp Val Val Pro His Ile Ser Ala Ala Gly 50 55 60 ArgGly Glu Ala Leu Glu Gly Ala Leu Gly Gln Gly Trp Asp Ser 65 70 75 Lys GluLys Glu Glu Ala Ala Ala Gly Glu His Ala Gly Gly Gln 80 85 90 Glu Phe GlyLeu Glu Gly Ser Ala Glu Glu Glu Val Thr Gly Arg 95 100 105 Gly Ser GlnVal Glu Ala Phe Glu Ser Arg Glu Gly Gly Pro Trp 110 115 120 Gly Gly ArgVal Glu Ala Glu Glu Ser Ala Gly Ala Glu Asp Ser 125 130 135 Cys Gly LeuAsp Pro Ala Gly Ser Gln Thr Ala Arg Ala Glu Gly 140 145 150 Met Gly AlaMet Val Glu Ala Gly Gly Leu Leu Glu Lys Trp Thr 155 160 165 Leu Leu GluGlu Glu Ala Val Gly Trp Gln Glu Arg Glu Gln Arg 170 175 180 Glu Asp SerGlu Gly Arg Cys Gly Asp Tyr His Pro Glu Gly Glu 185 190 195 Ala Pro ArgLeu Leu Asp Ala Glu Gly Leu Met Val Thr Gly 200 205 5 13 PRT artificialsequence UNSURE Amino acid sequence of a fragment from peptide 29 fromMBP protein; Xaa at position 1 is Glu, Leu or Ala; Xaa at position 2 isAla or Leu; Xaa at position 3 is Gln, Val or Glu 5 Xaa Xaa Xaa Ala GluGly Leu Met Val Thr Gly Gly Arg 5 10 6 7 PRT artificial sequence UNSUREAmino acid sequence of a fragment from peptide 18 from MBP protein; Xaaat position 1 is Val or Glu; Xaa at position 2 is Ala or Leu 6 Xaa XaaVal Met Gly Gln Met 5 7 751 DNA Homo sapien 7 gcggccgcgt ctaccgcggccgcgtctacg acagacagga tggacttcct ccggctatac 60 ctccctgggc tgcaccaggccttgaggggg gcactggatt ccctcggcac ctttgtctcc 120 tacctcctgg gagatgcagtccccactgta gagcgggagg cgcaggcggc tgaggaactg 180 ggggtggtgg cggtgggaaagacagggaag attgtagagg aggaagccca ggaggacctg 240 gagggcctta gaggcagccaaaacgagggg gctggaaggc tgagagggcc tggagatgac 300 agaagacatg aagtggggagctcagctgta gaacagacct ggggctgggg agatggcagc 360 tcccatgggt cccaagcagagaggcaggac agtggggctg gggagacagc caaggctgcc 420 aggtgccagg agccaagcgcccacttggag gccagaaaga aatccaaggc agggtctggg 480 gcttgccaag acaggagcggccaagcccag gagaggcagg agtcccatga gcaggaagtg 540 aacagagagg agaggctgagaagctgggaa caggaggagg aggaggaaga ggtcagggca 600 agggagccag ggatggccagaggggcggag tcagagtgga cctggcatgg ggagacggag 660 gggaaggctg gtgctgttgggccaaaggcg gcaggggaca accgggagat ggagcagggg 720 gtcagggagg cagatgcaggggaaactgag g 751 8 1466 DNA Homo sapien 8 cgggagatgg agcagggggtcagggaggca gatgcagggg aaactgagga gcctggggcc 60 gaaggggctg ggaaaggagaagaggtggta gtggtggaga aggcctgtga aagcactagg 120 gcatggggga cgtggggcccaggggcagag cctgaggact ggggaatctt aggcagagag 180 gaggccagga caaccccaggtagggaagag gccagggcaa ttttagatgg ggaggaagcc 240 aggacaatct caggcggggaggaggctgag acagcctcag gcggggagga ggctgaaaca 300 gcctcaggcg gggaggaggccgggacagcc tcgggagggg aggaggccgg gatatcctca 360 ggcggggagg ctgggacagcctcaggaggg gaggaggccg ggacagcctc tggaggggac 420 gaggcctgga caacctcaggcaaagaggag gctgacctgc tgggagtcag acagactcaa 480 tatggagcag ttccaggagaaaggctccta gaggctactg gaaaagtctg ggtcctagag 540 gaggaggggg atgaggagagagaggctgag gtgagccctt tccccaaaca ggcccaggtc 600 ctgggcactg aaagaacagaagaggctgct gagagccaga ccgcagggag ggaagctgtg 660 ggaggccagg aggcaggggagagctttgag ggccaggtag acctgcgtgg taaggaggct 720 gagatgaggc aggacttggggatcagggcc gaccgggcca agatggaaga gctggtacag 780 gcagaggagg cccaggaggagagagggagc agcagggatc cagtggctga gctgccctca 840 gatggagagg ctgaaggcactgccgacttg gaggcaactc cagaggccag gcctgaggag 900 gagctcacag gggaggagagtgaggcggcc cagactagct gtggcctact gggcgtggaa 960 tggggtggcc tcacacacagcgtcaccaaa ggccagggac ctgagctgat ggggggtgcc 1020 cagaccccaa ctaagcaacccgaggaaagg gaggcagggg aggtggagct catgggagtt 1080 ctggccctga gcaaagaggagcaggagagg agcctggagg caggtcccag gcacgcgggg 1140 tctgtaaagc ctgaggcctccgaggccttc ccaggagcct gggaaaaccg cacgagaaag 1200 gacatggaga gaggaaatactcaggaggat gcggccgatg gcgagcagcg ggaggaggag 1260 gagactgcgg gaggccagaccctggcggct gaggctgaag gagaccgaga gtctgaacta 1320 tcagaagtcc cagaggcaggcggggagggg ctgacaaccc aggacgcggg atgtggaact 1380 gaggagggag aggcatctgtctcagagaac caggagctgg acggaagcac aggggcagac 1440 gcagggcctt gcccgtcactgggaga 1466 9 1851 DNA Homo sapien 9 tcaggaggat gcggccgatg gcgagcagcgggaggaggag gagactgcgg gaggccagac 60 cctggcggct gaggctgaag gagaccgagagtctgaacta tcagaagtcc cagaggcagg 120 cggggagggg ctgacaaccc aggacgcgggatgtggaact gaggagggag aggcatctgt 180 ctcagagaac caggagctgg acggaagcacaggggcagac gcagggcctt gcccgtcact 240 gggagaggcc tatgccagag aaactgaggatgaggaggcg gaggctgaca gaacatccag 300 aagaggctgg aggctgcaag cggtggctgtgggcctcccg gaccgtgagg atgcacagac 360 tggctctgtg gctgctggga ttatggggggtgatgtggtc ccacacatca gcgctgctgg 420 cgctggtgaa gctttggaag gggcgcttgggcaaggctgg gactcgaaag aaaaggaaga 480 ggcagcagca ggagagcatg caggtgggcaagaatttggt ctggagggct cagcagagga 540 agaggtgact ggcagaggca gccaagtagaggcttttgag tccagggagg gaggaccttg 600 gggagggcgg gtagaggccg aggaatctgcaggcgcagag gacagctgtg ggctggatcc 660 cgcgggctcc cagacagcga gggcagaggggatgggagcc atggtggagg ctggggggct 720 tctagaaaag tggacgctgt tggaagaagaggctgttgga tggcaggaga gagaacagag 780 ggaagacagt gaggggcggt gtggggactaccaccctgag ggagaggcac caaggctcct 840 tgatgcagag ggtctcatgg tgaccgggggccggagggca gaggccaagg agactgagcc 900 agaaagcctg gaacatgtca ggggccaggaggagcagcca acacaccagg cccctgcaga 960 agctgcgccg gagtcagtcg gggaagccgagacggctgag gccatgggca gtgccagagg 1020 aggtgctgcc aacagctgga gcgaggccccgctccccggg tccctcctag acgtctctgt 1080 cccaaggagt cgcgtgcacc tctcgagaagctcctcacag cgtcgctccc ggccctcttt 1140 tcgtcggact ccggcctggg agcagcaggaggagccccca gcccccaacc ctcctgagga 1200 ggagctgtca gctcctgagc agagacccctccagctggag gaacccctgg agccaagccc 1260 tctgaggcat gatgggaccc cggtgccagccaggagaagg cccctgggac acgggtttgg 1320 cctcgcgcac cctggcatga tgcaggagctgcaagcccgt ctgggccggc ctaagcccca 1380 gtgactgaga cccggtgctc tgggagccaggccctgagtg ggtgccagaa ggcttgctcc 1440 aatgccactg agccctgctc cctctgccactgtggacaca tcctctccac cctctgggcc 1500 tcagtgtctt gatgtatcat tcatggagcaggcaaaacca gacgtctggg aataccgtga 1560 acttaaggag tctgattctc cgacacaggctggtggacca cctaccccac tgagaccacc 1620 tctcagggtg cctgccctgg ttcctccccagcctgagtca gctgtctgga ctgcaaggag 1680 gctgggcacg ggggctcacg cctgtcaccccagagctttg ggaggccaag gtgggaggat 1740 cgcttgagac caggagttcg agaccagcctgggcagcata gcaagatccc catcttttaa 1800 aaacaaaata aaacaataaa gactgcaaggaaaaaaaaaa aaaaaaaaaa a 1851 10 11 PRT artificial sequenceMOD_RES/AMIDATION Amino acid sequence of a fragment from peptide 29 fromMBP protein; C-terminal arginine was amidated at position 11 10 Cys AlaGlu Gly Leu Met Val Thr Gly Gly Arg 5 10 11 10 PRT Artificial SequencePeptide previously used to produce receptor-specific antibodies 11 AlaGlu Gly Leu Met Val Thr Gly Gly Arg 5 10 12 19 DNA Artificial Sequence2048-fold degenerate oligonucleotide corresponding to base pairs2763-2745 of apoB48R cDNA sequence; n = a,t,c, or g at positions 3, 6,9, 15, and 18 12 ccnccngtna ccatnarnc 19 13 17 DNA Artificial Sequence256-fold degenerate oligonucleotide corresponding to base pairs2754-2738 of apoB48R cDNA sequence; n = a, t, c, or g at positions 6, 9,and 15 13 accatnarnc cytcngc 17 14 21 DNA Artificial Sequence (gt10forward primer 14 agcaagttca gcctggttaa g 21 15 17 DNA ArtificialSequence Primer based on the sequence of the pcr-137 PCR product (bp2763-2745 of ApoB48R cDNA) 15 ccccccgtca ccatgag 17 16 17 DNA ArtificialSequence Primer based on the sequence of the pcr-137 PCR product (bp2754-2738 of ApoB48R cDNA) 16 accatgagrc cctctgc 17 17 17 DNA ArtificialSequence Antisense primers were based on the 5′-end sequence of theTHP-1 (73 clone (bp 2203-2187 of ApoB48R cDNA) 17 ccagcctctt ctggatg 1718 17 DNA Artificial Sequence Antisense primers were based on the 5′-endsequence of the THP-1 (73 clone (bp 2139-2123 of ApoB48R cDNA) 18tctcccagtg acgggca 17 19 24 DNA Artificial Sequence Antisense primersbased on the 5′ end sequence of clone pcr-4-3 (bp 722-699) 19 cctcagtttcccctgcatct gcct 24 20 360 DNA Homo sapiens Intron 1 of ApoB48R GenomicDNA, between bp 67 and 68 of the cDNA 20 gtgagaaggg cagacagctgccagatactt gcaccccatt ccctggggcc tcacttccgg 60 gcacctcccc tggggcctcacctttcccct cctccttctg atctcctcta actggagatt 120 gctttctcag gttcaggcagactcctggcc taatattttc tgaatttcag tccccacctc 180 caaccatgcg tcctcgtacccctaatcgat gccccttctg gctccttctg caaatcctct 240 tcttctcctt tcagatcccagtaccctctt ccttaacctg ggctcctcca gccagggccc 300 ccagggaaag ggctgggactctcctcaatg actctcccct ctctctctct tttttcctag 360 21 84 DNA Homo sapiensIntron 2 of ApoB48R Genomic DNA (between bp 3228 and 3229 of the cDNA)21 gtgagggctc ttggtggggt ctcgggggga acgagtggaa tcccgaagcc ggccccatgg 60tcctctgtgc cccctttcct gcag 84 22 84 DNA Homo sapiens Intron 3 of ApoB48RGenomic DNA (between bp 3207 and 3208 of the cDNA) 22 gtgagggctcttggtggggt ctcgggggga acgagtggaa tcccgaagcc ggccccatgg 60 tcctctgtgccccctttcct gcag 84

What is claimed is:
 1. An isolated DNA molecule encoding amonocyte-macrophage cell surface apoB48 receptor protein (apoB48R), saidDNA molecule selected from the group consisting of: (a) a DNA moleculecomprising SEQ ID No. 1 encoding said monocyte-macrophage cell surfaceapoB48 receptor protein as set forth in SEQ ID No. 2 or a fragmentthereof; and, (b) a DNA molecule differing from the DNA molecule of (a)in codon sequence due to the degeneracy of the genetic code, and whichencodes said monocyte-macrophage cell surface apoB48 receptor protein asset forth in SEQ ID No. 2 or a fragment thereof.
 2. A vector containingthe DNA molecule of claim 1 and regulatory elements necessary forexpression of said DNA in a cell, said vector adapted for expression ina recombinant cell.
 3. A host cell containing the vector of claim
 2. 4.An isolated DNA molecule encoding a portion of the apoB48 receptorprotein, said DNA molecule selected from the group consisting of SEQ IDNo. 3, SEQ ID No. 7, SEQ ID No. 8 and SEQ ID No.
 9. 5. A vectorcontaining the isolated DNA molecule of claim 4 and regulatory elementsnecessary for expression of said DNA in a cell, said vector adapted forexpression in a recombinant cell.
 6. A host cell containing the vectorof claim
 5. 7. An isolated monocyte-macrophage cell surface apoB48receptor protein (apoB48R) having the sequence SEQ ID No.
 2. 8. Anantibody directed to an isolated monocyte-macrophage cell surface apoB48receptor protein having the sequence encoded by a DNA molecule selectedfrom the group consisting of SEQ ID No. 1, SEQ ID No. 3, SEQ ID No. 7,SEQ ID No. 8, SEQ ID No. 9 and a fragment thereof.
 9. A method ofcell-specific delivery of a therapeutic compound to an individual inneed of such treatment, comprising the steps of: providing a recognitioncompound selected from the group consisting of a peptide, an antibodyand an antibody fragment having the ability to bind to themonocyte-macrophage cell surface apoB48 receptor protein of claim 7 (SEQID No. 2), or a portion of said sequence; and incorporating saidrecognition compound into a delivery vehicle containing said therapeuticcompound.
 10. The method of claim 9, wherein said delivery vehicle is aliposome.
 11. The method of claim 9, wherein said therapeutic compoundis selected from the group consisting of cytocidal drugs, cytotoxicdrugs, genes, vitamins, hormones, cytokines, growth factors and growthinhibitors.
 12. The method of claim 9, wherein said cell is selectedfrom the group consisting of monocytes, macrophages, endothelial cells,placental cells, peripheral blood leukocytes, bone marrow cells,astrocytes, osteoclasts and other monocyte-derived cells.
 13. The methodof claim 9, wherein said individual has a disease selected from thegroup consisting of monocytic leukemia, tuberculosis, leprosy, and AIDS.14. The method of claim 9, wherein said therapeutic compound deliveredto said individual has an effect selected from the group consisting ofinhibition of angiogenesis, enhancement of angiogenesis, inhibition offibrinolysis, enhancement of fibrinolysis, inhibition of tissue factorproduction and enhancement of tissue factor production.
 15. The methodof claim 9, wherein said therapeutic compound is delivered to adeveloping embryo in said individual.
 16. The method of claim 9, whereinsaid therapeutic compound is a label used to locate atheroscleroticplaques.
 17. The method of claim 9, wherein said therapeutic compound isa compound used to eliminate atherosclerotic plaques.
 18. A method ofinhibiting the binding of lipoproteins to cells, comprising the step oftreating said cells with an agent which binds a monocyte-macrophage cellsurface apoB48 receptor protein of claim 7 (SEQ ID No. 2), wherein saidbinding inhibits the binding of lipoproteins to cells, therebyinhibiting foam cell formation and increased monocyte adhesion toendothelial cells.
 19. The method of claim 18, wherein said agent isselected from the group consisting of a peptide, an antibody directed toan isolated apoB48 receptor protein having the sequence encoded by a DNAmolecule selected from the group consisting of SEQ ID No. 1, SEQ ID No.3, SEQ ID No. 7, SEQ ID No. 8, SEQ ID No. 9 and a fragment thereof. 20.The method of claim 19, wherein said peptide is lipoprotein lipase or afragment thereof.
 21. The method of claim 18, wherein said cells areselected from the group consisting of monocytes, macrophages,endothelial cells, placental cells, peripheral blood leukocytes, bonemarrow cells, astrocytes, osteoclasts and other monocyte-derived cells.22. A method of enhancing hepatic uptake and catabolism ofapoB48-containing lipoproteins in an individual in need of suchtreatment, comprising the step of administering to said individual aneffective amount of a vector encoding the monocyte-macrophage cellsurface apoB48 receptor protein of claim 7 (SEQ ID No. 2), or a portionof said protein.
 23. The method of claim 22, wherein said individual hasbeen diagnosed with a disease selected from the group consisting ofPattern B phenotype, familial combined hyperlipidemia, familialhypercholesterolemia, non-familial hypercholesterolemia,hypertriglyceridemia and low plasma high density lipoprotein levels. 24.A method of evaluating an individual at risk for cardiovascular disease,comprising the steps of: (a) extracting a sample ofmonocytes-macrophages and triglyceride-rich lipoproteins from plasma ofsaid individual and from a control individual not considered at risk forcardiovascular disease; and (b) comparing the binding affinity (K_(d))of the apoB48 receptor of claim 7 between said individual at risk andsaid control individual, whereby a difference in said binding affinitybetween said individual at risk and said control individual isindicative of an alteration in either or both the apoB cell-surfacereceptor protein an d triglyceride-rich lipoproteins, wherein saidalteration in said apoB cell-surface receptor protein ortriglyceride-rich lipoproteins is indicative of dyslipidemias, abnormalpostprandial triglyceride metabolism or Pattern B phenotype in saidindividual at risk.
 25. A method of evaluating an individual at risk forcardiovascular disease, comprising the steps of: (a) extracting a sampleof monocytes-macrophages from plasma of said individual; and (b)comparing the binding affinity (K_(d)) of the apoB48 receptor of claim 7of said individual at risk and the apoB48 receptor of control THP-1cells for triglyceride-rich lipoproteins, whereby a difference in saidbinding affinity between said individual at risk and said control cellsis indicative of an alteration in the apoB cell-surface receptor proteinin said individual at risk, wherein said alteration in said apoBcell-surface receptor protein is indicative of dyslipidemias, abnormalpostprandial triglyceride metabolism or Pattern B phenotype in saidindividual at risk.
 26. A method of evaluating an individual at risk forcardiovascular disease, comprising the steps of: (a) extracting a sampleof monocytes-macrophages and triglyceride-rich lipoproteins from plasmaof said individual; and (b) comparing the binding affinity (K_(d)) ofsaid triglyceride-rich lipoproteins for the apoB receptor of claim 7said individual at risk and the apoB receptor of THP-1 control cells,whereby a difference in said binding affinity between said individual atrisk and said control cells is indicative of an alteration in thetriglyceride-rich lipoproteins, wherein said alteration in saidtriglyceride-rich lipoproteins is indicative of dyslipidemias, abnormalpostprandial triglyceride metabolism or Pattern B phenotype in saidindividual at risk.
 27. A method of evaluating an individual at risk forcardiovascular disease, comprising the steps of: (a) extracting a sampleof monocytes-macrophages from plasma of said individual; and (b)performing a Western blot analysis on proteins of saidmonocytes-macrophages using an antibody directed towards the protein ofclaim 7 (SEQ ID No. 2), or fragments thereof, whereby a difference inthe migration or mobility of said proteins between said individual atrisk and said control is indicative of an alteration in the apoBcell-surface receptor protein, wherein said alteration in said apoBcell-surface receptor protein is indicative of dyslipidemias, abnormalpostprandial triglyceride metabolism or Pattern B phenotype in saidindividual at risk.
 28. The method of claim 27, wherein said control isselected from the group consisting of a THP-1 clonal cell line or asample of monocytes-macrophages from plasma of a control individual notconsidered at risk for cardiovascular disease.
 29. A method ofevaluating an individual at risk for cardiovascular disease, comprisingthe steps of: (a) extracting a sample of monocytes-macrophages fromplasma of said individual; and (b) isolating and analyzing RNAs of saidmonocytes-macrophages, whereby a difference in said RNAs between saidindividual at risk and said control is indicative of an alteration inthe apoB cell-surface receptor protein, wherein said alteration in saidapoB cell-surface receptor protein is indicative of dyslipidemias,abnormal postprandial triglyceride metabolism or Pattern B phenotype insaid individual at risk.
 30. The method of claim 29, wherein saidanalyzing is performed by Northern blot analysis using a DNA probeselected from the group consisting of SEQ ID No. 1, SEQ ID No. 3, SEQ IDNo. 7, SEQ ID No. 8 and SEQ ID No. 9, or fragments thereof.
 31. Themethod of claim 29, wherein said analyzing is performed by RT-PCR usingoligonucleotides complementary to SEQ ID No.
 1. 32. The method of claim29, wherein said control is selected from the group consisting of aTHP-1 clonal cell line or a sample of monocytes-macrophages from plasmaof a control individual not considered at risk for cardiovasculardisease.
 33. A method of evaluating an individual at risk forcardiovascular disease, comprising the steps of: (a) extracting a sampleof monocytes-macrophages from plasma of said individual; and (b)comparing the number of apoB receptors on said monocytes-macrophagesbetween said individual at risk and said control, whereby a differencein said number of receptors between said individual at risk and saidcontrol is indicative of an alteration in the apoB cell-surface receptorprotein, wherein said alteration in said apoB cell-surface receptorprotein is indicative of dyslipidemias, abnormal postprandialtriglyceride metabolism or Pattern B phenotype in said individual atrisk.
 34. The method of claim 33, wherein said control is selected fromthe group consisting of a THP-1 clonal cell line or a sample ofmonocytes-macrophages from plasma of a control individual not consideredat risk for cardiovascular disease.